AP2 domain transcription factor ODP2 (Ovule Development Protein 2) and methods of use

ABSTRACT

Methods and compositions for modulating plant development are provided. Nucleotide sequences and amino acid sequences encoding Ovule Development Protein 2 (ODP2) proteins are provided. The sequences can be used in a variety of methods including modulating development, developmental pathways, altering oil content in a plant, increasing transformation efficiencies, modulating stress tolerance, and modulating the regenerative capacity of a plant. Transformed plants, plant cells, tissues, and seed are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/131,181 filed Apr. 18, 2016, now U.S. Pat. No. 10,113,175, which is aDivision of U.S. application Ser. No. 13/790,641, filed Mar. 8, 2013,now U.S. Pat. No. 9,340,796, which is a continuation of U.S. applicationSer. No. 12/503,482, filed on Jul. 15, 2009, now U.S. Pat. No.8,420,893, which is a continuation of U.S. application Ser. No.11/045,802, filed on Jan. 28, 2005, now U.S. Pat. No. 7,579,529, whichclaims priority to U.S. Provisional Application No. 60/541,122, filed onFeb. 2, 2004, all of which are hereby incorporated by reference in theirentireties.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named1759USCNT5 SequenceListing created on Sep. 26, 2018 and having a size of146 kilobytes and is filed concurrently with the specification. Thesequence listing contained in this ASCII formatted document is part ofthe specification and is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The invention relates to the field of the genetic manipulation ofplants, particularly the modulation of gene activity and development inplants.

BACKGROUND OF THE INVENTION

Cell division plays a crucial role during all phases of plantdevelopment. The continuation of organogenesis and growth responses to achanging environment requires precise spatial, temporal anddevelopmental regulation of cell division activity in meristems. Suchcontrol of cell division is also important in organs themselves forexample, leaf expansion, and secondary growth. A complex networkcontrols cell proliferation in eukaryotes. Various regulatory pathwayscommunicate environmental constraints, such as nutrient availability,mitogenic signals such as growth factors or hormones, or developmentalcues such as the transition from vegetative to reproductive. Ultimately,these regulatory pathways control the timing, frequency (rate), planeand position of cell divisions. The regulation of cell division impactsa variety of developmental pathways including transformation and plantregeneration.

Current transformation technology provides an opportunity to engineerplants with desired traits. Major advances in plant transformation haveoccurred over the last few years. However, in many major crop plants,serious genotype limitations still exist. Transformation of someagronomically important crop plants continues to be both difficult andtime consuming.

For example, it is difficult to obtain a culture response from somemaize genotypes. Typically, a suitable culture response has beenobtained by optimizing medium components and/or explant material andsource. This has led to success in some genotypes. While, transformationof model genotypes is efficient, the process of introgressing transgenesinto production inbreds is laborious, expensive and time consuming. Itwould save considerable time and money if genes could be moreefficiently introduced into and evaluated directly into inbreds.Accordingly, methods are needed in the art to increase transformationefficiencies of plants.

Influencing cell cycle and cell division can also affect variousdevelopmental pathways in a plant. Pathways of interest include thosethat influence embryo development. The AP2/ERF family of proteins is aplant-specific class of putative transcription factors that have beenshown to regulate a wide-variety of developmental processes and arecharacterized by the presence of a AP2/ERF DNA binding domain. TheAP2/ERF proteins have been subdivided into two distinct subfamiliesbased on whether they contain one (ERF subfamily) or two (AP2 subfamily)DNA binding domains.

One member of the AP2 family that has been implicated in a variety ofcritical plant cellular functions is the Baby Boom protein (BBM). TheBBM protein from Arabidopsis is preferentially expressed in seed and hasbeen shown to play a central role in regulating embryo-specificpathways. Overexpression of BBM has been shown to induce spontaneousformation of somatic embryos and cotyledon-like structures on seedlings.See, Boutiler et al. (2002) The Plant Cell 14:1737-1749. Thus, membersof the AP2 protein family promote cell proliferation and morphogenesisduring embryogenesis. Such activity finds potential use in promotingapomixis in plants.

Apomixis refers to the production of a seed from the maternal ovuletissue in the absence of egg cell fertilization (Koltunow (1995) PlantPhysiol 108:1345-1352). Apomixis is a valuable trait for cropimprovement since apomictic seeds give rise to clonal offspring and cantherefore be used to genetically fix hybrid lines. The production ofhybrid lines is intensive and costly. Production of seed throughapomixis avoids these problems in that once a hybrid has been produced,it can be maintained clonally, thereby eliminating the need to maintainand cross separate parent lines. The use of apomictic seeds alsoeliminates the use of cuttings or tissue culture techniques to propagatelines, reduces the spread of disease which are easily spread throughvegetative-propagated tissues and in many species, reduces the size ofthe propagule leading to lower shipping and planting costs. Methods aretherefore needed for the efficient production of apomictic seed.

Members of the APETALA2 (AP2) family of proteins play critical roles ina variety of important biological events including development, plantregeneration, cell division, etc. Accordingly, it is valuable to thefield of agronomic development to identify and characterize novel AP2family members and develop novel methods to modulate embryogenesis,transformation efficiencies, oil content, starch content and yield in aplant.

BRIEF SUMMARY OF THE INVENTION

Methods and compositions are provided to modulate plant developmentusing DNA, RNA or protein derived from the maize AP2 family memberZmODP2. The present invention provides an isolated polypeptidecomprising an amino acid sequence selected from the group consisting of:(a) the polypeptide comprising the amino acid sequence of SEQ ID NO:2,26, or 28; (b) the polypeptide having at least 50%, sequence identity toSEQ ID NO:2, 26, or 28, wherein the polypeptide has Ovule DevelopmentProtein 2 (ODP2) activity; (c) the polypeptide encoded by apolynucleotide that hybridizes under stringent conditions to apolynucleotide comprising the complement of SEQ ID NOS:1, 3, 25, or 27,wherein the stringent conditions comprise hybridization in 50%formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C.to 65° C.; and, (d) the polypeptide having at least 70 consecutive aminoacids of SEQ ID NO:2, 26, or 28, wherein the polypeptide retains ODP2activity.

Further compositions of the invention include an isolated polynucleotideselected from the group consisting of: (a) the polynucleotide comprisingSEQ ID NO:1, 3, 25 or 27; (b) the polynucleotide encoding the amino acidsequence of SEQ ID NO:2, 26 or 28; (c) the polynucleotide having atleast 50% sequence identity to SEQ ID NO:1, 3, 25 or 27, wherein thepolynucleotide encodes a polypeptide having ODP2 activity; (d) thepolynucleotide having at least 200 consecutive nucleotides of SEQ IDNO:1, 3, 25 or 27 or a complement thereof; and, (e) the polynucleotidethat hybridizes under stringent conditions to the complement of thepolynucleotide of (a), wherein the stringent conditions comprisehybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a washin 0.1×SSC at 60° C. to 65° C. Nucleotide constructs comprising thepolynucleotide of the invention are also provided.

Additional compositions of the invention include plants having aheterologous polynucleotide of the invention operably linked to apromoter that drives expression in the plant. The plant can be a plantcell, a plant part, a seed, or a grain. Methods are provided to modulatedevelopment in a plant. In one embodiment, the plant of the inventionhas an altered oil phenotype. In specific embodiments the oil content ofthe plant is decreased. In other embodiments, starch production of theplant is modified. In specific embodiments, the starch content of theplant is increased. In another embodiment, the regenerative capacity ofthe plant is modified. In yet another embodiment, the plant produces anasexually derived embryo. In still another embodiment, thetransformation efficiency of the plant is increased. In anotherembodiment, the seed set is increased or maintained during periods ofabiotic stress. In still another embodiment, haploid embryos areproduced from male or female gametes.

Methods of the invention comprise methods for modulating the activityand/or level of a polypeptide in a plant. This method comprisesproviding to the plant an ODP2 sequence of the invention.

The present invention further provides a method for altering the oilphenotype in a plant. The method comprises providing to the plant anODP2 sequence of the invention; and, thereby altering the oil phenotypeof the plant.

The present invention further provides a method for modifying starchproduction in a plant. The method comprises providing to the plant anODP2 sequence of the invention; and, thereby modifying starch productionof the plant.

The present invention further provides a method for producing asexuallyderived embryos. The method comprises introducing into a plant ODP2sequence of the present invention; and, thereby producing asexuallyderived embryos. The asexually derived embryos can be somatic embryos,adventitious embryos, or gametophytic embryos.

The present invention also provides a method for modifying theregenerative capacity of a plant. The method comprises introducing intothe plant an ODP2 nucleotide sequence of the invention, and therebymodifying the regenerative capacity of the plant.

The present invention also provides a method of transforming a plant.The method comprises providing to target plant an ODP2 sequence of theinvention, and, transforming into the target plant a nucleotide sequenceof interest. The regenerative capacity can be modified to includetissues normally not amenable to culture including but not limited toleaves, stems, and mature seed.

The invention further provides a method for increasing transformationefficiency in a plant. The method comprises providing to the plant anODP2 nucleotide sequence of the invention, and thereby increasing thetransformation efficiency of the plant.

The invention further provides a method for increasing or maintainingyield in a plant under abiotic stress. The method comprises providing tothe plant an ODP2 nucleotide sequence of the invention, and therebyincreasing the stress tolerance of the plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment of the amino acid sequence of maize OvuleDevelopment Protein 2 (Zm-ODP2) (SEQ ID NO:2) with OsAnt (Accession No.BAB89946; SEQ ID NO:26), OSBNM (Accession No. AAL47205; SEQ ID NO:28);OSODP (Accession No. CAEO5555; SEQ ID NO:29); AtODP (NP_197245; SEQ IDNO:30); ATBBM (Accession No. AAM33803; SEQ ID NO:31); BnBBM1 (AAM33800;SEQ ID NO:32); BnBBM2 (Accession No. AAM33801; SEQ ID NO:33); ATODP(Accession No. NP_175530; SEQ ID NO:34); AtODP (Accession No. BAB02492;SEQ ID NO:35); AtODP (Accession No. AAD30633; SEQ ID NO:36). All 11proteins present in the alignment have two AP2 (APETALA2; pfam00847.8)domains. Using the amino acid numbering of the Zm-ODP2 polypeptide, thefirst AP2 domain is from about amino acid 273 to about 343 and thesecond AP2 domain is from about amino acid 375 to about 437. A consensussequence for all 11 aligned polypeptides is also provided (SEQ IDNO:37).

FIG. 2 provides an amino acid alignment of the Zm-ODP2 amino acidsequence (ZM-ODP2_unmodifiedPEP; SEQ ID NO:2) with four polypeptidevariants of the Zm-ODP2 sequence. The variant amino acid sequencesinclude ZM-ODP2_modifiedPEP_id_97.3 (SEQ ID NO:20) which shares 97.3%amino acid sequence identity with SEQ ID NO:2;ZM-ODP2_modifiedPEP_id_92.4 (SEQ ID NO:21) which shares 92.4% amino acidsequence identity with SEQ ID NO:2; ZM-ODP2_modifiedPEP_id_87.3 (SEQ IDNO:22) which shares 87.3% amino acid sequence identity with SEQ ID NO:2;and, ZM-ODP2_modifiedPEP_id_82.4 (SEQ ID NO:23) which shares 82.4% aminoacid sequence identity with SEQ ID NO:2. The consensus sequence is setforth in SEQ ID NO:24.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the invention are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

COMPOSITIONS

Compositions of the invention include polynucleotide sequence and aminoacid sequence of Ovule Development Protein 2 (ODP2) proteins that areinvolved in regulating plant growth and development. In particular, thepresent invention provides for isolated nucleic acid moleculescomprising nucleotide sequences encoding the amino acid sequences shownin SEQ ID NO:2, 26, or 28. Further provided are polypeptides having anamino acid sequence encoded by a nucleic acid molecule (SEQ ID NO: 1, 3,25, or 27) described herein, and fragments and variants thereof.

The ODP2 polypeptides of the invention contain two predicted APETALA2(AP2) domains and are members of the AP2 protein family (PFAM AccessionPF00847). The AP2 domains of the maize ODP2 polypeptide are located fromabout amino acids 5273 to N343 and from about 5375 to R437 of SEQ IDNO:2). The AP2 family of putative transcription factors have been shownto regulate a wide range of developmental processes, and the familymembers are characterized by the presence of an AP2 DNA binding domain.This conserved core is predicted to form an amphipathic alpha helix thatbinds DNA. The AP2 domain was first identified in APETALA2, anArabidopsis protein that regulates meristem identity, floral organspecification, seed coat development, and floral homeotic geneexpression. The AP2 domain has now been found in a variety of proteins.

The ODP2 polypeptides of the invention share homology with severalpolypeptides within the AP2 family. FIG. 1 provides an alignment of themaize and rice ODP2 polypeptides of the present invention with 8 otherproteins having two AP2 domains. A consensus sequence of all proteinsappearing in the alignment is also provided in FIG. 1. The alignment ofFIG. 1 was generated using Align X® which employs a modified Clustal Walgorithm to generate multiple sequence alignments. FIG. 1 demonstratesthat the maize ODP2 polypeptide of the present invention (SEQ ID NO:2)shares about 51.7% sequence identity and 62.3% sequence similarityacross the full sequence with the rice sequences of OsBNM3 (ovuledevelopment aintegumenta-like protein) (Genbank Accession No. AAL47205;SEQ ID NO:28). In addition, the ODP2 polypeptide of SEQ ID NO:2 shares65.4% sequence identity and 72.7% sequence similarity across the fullsequence to a putative ovule development protein from rice (OS) (GenbankAccession No. BAB89946; SEQ ID NO:26).

The OsBNM3 polypeptide sequence (SEQ ID NO:28), the OS polypeptide (SEQID NO:26), as well as the ODP2 sequence (SEQ ID NO:2) share homologywith Arabidopsis Baby Boom (AtBBM, AAM33803; SEQ ID NO:31). Blastalignments demonstrate that Zm-ODP2 shares about 38.1% sequence identityand about 46.3% sequence similarity across the full length of theArabidopsis Baby Boom polypeptide (AtBBM). See FIG. 1. The AtBBMpolypeptide encodes an AP2 domain transcription factor and is optimallyexpressed in the developing embryo and seeds. AtBBM has been shown totrigger formation of somatic embryos and cotyledon-like structures onseedlings and thus activates signal transduction pathways leading to theinduction of embryo development from differentiated somatic cells. See,for example, Boutiler et al. (2002) Plant Cell 14:1737-49), hereinincorporated by reference. Accordingly, the ODP2 sequences of thepresent invention also find use in modifying the regenerativecapabilities of plants and rendering the plant embryogenic.

In addition, other polypeptides that influence ovule and embryodevelopment and stimulate cell growth, such as, Lec1, Kn1 family,WUSCHEL, Zwille, and Aintegumeta (ANT) allow for increasedtransformation efficiencies when expressed in plants. See, for example,U.S. Application No. 2003/0135889, herein incorporated by reference. Infact, a maize Lec1 homologue of the Arabidopsis embryogenesiscontrolling gene AtLEC1, has been shown to increase oil content andtransformation efficiencies in plants. See, for example, WO 03001902 andU.S. Pat. No. 6,512,165. Accordingly, the Zm-ODP2 sequences of theinvention find further use in increasing transformation efficiencies inplants.

The invention encompasses isolated or substantially purified nucleicacid or protein compositions. An “isolated” or “purified” nucleic acidmolecule or protein, or biologically active portion thereof, issubstantially or essentially free from components that normallyaccompany or interact with the nucleic acid molecule or protein as foundin its naturally occurring environment. Thus, an isolated or purifiednucleic acid molecule or protein is substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. Optimally, an “isolated” nucleic acid is free ofsequences (optimally protein encoding sequences) that naturally flankthe nucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolatednucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived. A protein that is substantially free ofcellular material includes preparations of protein having less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein.When the protein of the invention or biologically active portion thereofis recombinantly produced, optimally culture medium represents less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors ornon-protein-of-interest chemicals.

Fragments and variants of the disclosed nucleotide sequences andproteins encoded thereby are also encompassed by the present invention.By “fragment” is intended a portion of the nucleotide sequence or aportion of the amino acid sequence and hence protein encoded thereby.Fragments of a nucleotide sequence may encode protein fragments thatretain the biological activity of the native protein and hence have ODP2activity. Alternatively, fragments of a nucleotide sequence that areuseful as hybridization probes generally do not encode fragment proteinsretaining biological activity. Thus, fragments of a nucleotide sequencemay range from at least about 20 nucleotides, about 50 nucleotides,about 100 nucleotides, and up to the full-length nucleotide sequenceencoding the proteins of the invention.

By “ODP2 activity” or “Ovule Development Protein 2 activity” is intendedthe ODP2 polypeptide has at least one of the following exemplaryactivities: increases the regenerative capability of a plant cell,renders the plant cell embryogenic, increases the transformationefficiencies of a plant cell, alters the oil content of a plant cell,binds DNA, increases abiotic stress tolerance, increases or maintainsyield under abiotic stress, increases asexual embryo formation, altersstarch content, alters embryo size or activates transcription. Methodsto assay for such activity are known in the art and are described morefully below.

A fragment of an ODP2 nucleotide sequence that encodes a biologicallyactive portion of an ODP2 protein of the invention will encode at least15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 709 contiguous amino acids, or up to the total number of aminoacids present in a full-length ODP2 protein of the invention (forexample, 710 amino acids for SEQ ID NO: 2, 692 amino acids for SEQ IDNO: 25 and 597 for SEQ ID NO:27). Fragments of an ODP2 nucleotidesequence that are useful as hybridization probes or PCR primersgenerally need not encode a biologically active portion of an ODP2protein.

Thus, a fragment of an ODP2 nucleotide sequence may encode abiologically active portion of an ODP2 protein, or it may be a fragmentthat can be used as a hybridization probe or PCR primer using methodsdisclosed below. A biologically active portion of an ODP2 protein can beprepared by isolating a portion of one of the ODP2 nucleotide sequencesof the invention, expressing the encoded portion of the ODP2 protein(e.g., by recombinant expression in vitro), and assessing the activityof the encoded portion of the ODP2 protein. Nucleic acid molecules thatare fragments of an ODP2 nucleotide sequence comprise at least 16, 20,50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,800, 900, 1,000, 1,100, 1,200, 1,300, or 1,400, 1,500, 1,600, 1,700,1,800, 1,900, 2,000, 2,100, 2,200 contiguous nucleotides, or up to thenumber of nucleotides present in a full-length ODP2 nucleotide sequencedisclosed herein (for example, 2,260, 2133, 2079, and 1794 nucleotidesfor SEQ ID NOS:1, 3, 25 and 27, respectively).

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more internal sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. As used herein, a “native”polynucleotide or polypeptide comprises a naturally occurring nucleotidesequence or amino acid sequence, respectively. For polynucleotides,conservative variants include those sequences that, because of thedegeneracy of the genetic code, encode the amino acid sequence of one ofthe ODP2 polypeptides of the invention. Naturally occurring variantssuch as these can be identified with the use of well-known molecularbiology techniques, as, for example, with polymerase chain reaction(PCR) and hybridization techniques as outlined below. Variantpolynucleotides also include synthetically derived polynucleotide, suchas those generated, for example, by using site-directed mutagenesis butwhich still encode an ODP2 protein of the invention. Generally, variantsof a particular polynucleotide of the invention will have at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thatparticular polynucleotide as determined by sequence alignment programsand parameters described elsewhere herein.

Variants of a particular polynucleotide of the invention (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Thus, for example, an isolated polynucleotide thatencodes a polypeptide with a given percent sequence identity to thepolypeptide of SEQ ID NO:2, 26, or 28 are disclosed. Percent sequenceidentity between any two polypeptides can be calculated using sequencealignment programs and parameters described elsewhere herein. Where anygiven pair of polynucleotides of the invention is evaluated bycomparison of the percent sequence identity shared by the twopolypeptides they encode, the percent sequence identity between the twoencoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity.

“Variant” protein is intended to mean a protein derived from the nativeprotein by deletion or addition of one or more amino acids at one ormore internal sites in the native protein and/or substitution of one ormore amino acids at one or more sites in the native protein. Variantproteins encompassed by the present invention are biologically active,that is they continue to possess the desired biological activity of thenative protein, that is, the polypeptide has ODP2 activity (i.e.,modulating the regenerative capability of a plant, rendering the plantembryogenic, increasing the transformation efficiency of a plant,altering oil content of a plant, increasing cell proliferation,increasing abiotic stress tolerance, increasing or maintaining yieldunder abiotic stress, modifying starch content, increasing asexualembryo formation, binding DNA or regulating transcription) as describedherein. Such variants may result from, for example, genetic polymorphismor from human manipulation. Biologically active variants of a nativeODP2 protein of the invention will have at least about 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to the amino acid sequence forthe native protein as determined by sequence alignment programs andparameters described elsewhere herein. A biologically active variant ofa protein of the invention may differ from that protein by as few as1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, asfew as 4, 3, 2, or even 1 amino acid residue.

The proteins of the invention may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of the ODP2 proteins can beprepared by mutations in the DNA. Methods for mutagenesis and nucleotidesequence alterations are well known in the art. See, for example, Kunkel(1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987)Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker andGaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York) and the references cited therein. Guidanceas to appropriate amino acid substitutions that do not affect biologicalactivity of the protein of interest may be found in the model of Dayhoffet al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed.Res. Found., Washington, D.C.), herein incorporated by reference.Conservative substitutions, such as exchanging one amino acid withanother having similar properties, may be optimal.

Thus, the genes and nucleotide sequences of the invention include boththe naturally occurring sequences as well as mutant forms. Likewise, theproteins of the invention encompass both naturally occurring proteins aswell as variations and modified forms thereof. Such variants willcontinue to possess the desired ODP2 activity. Obviously, the mutationsthat will be made in the DNA encoding the variant must not place thesequence out of reading frame and optimally will not createcomplementary regions that could produce secondary mRNA structure. See,EP Patent Application Publication No. 75,444.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays. Various methods for screeningfor ODP2 activity are discussed in detail elsewhere herein.

Variant nucleotide sequences and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different ODP2 codingsequences can be manipulated to create a new ODP2 possessing the desiredproperties. In this manner, libraries of recombinant polynucleotides aregenerated from a population of related sequence polynucleotidescomprising sequence regions that have substantial sequence identity andcan be homologously recombined in vitro or in vivo. For example, usingthis approach, sequence motifs encoding a domain of interest may beshuffled between the ODP2 gene of the invention and other known ODP2genes to obtain a new gene coding for a protein with an improvedproperty of interest, such as an increased K_(m) in the case of anenzyme. Strategies for such DNA shuffling are known in the art. See, forexample, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech.15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al.(1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998)Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

The nucleotide sequences of the invention can be used to isolatecorresponding sequences from other organisms, particularly other plantsincluding other monocots. In this manner, methods such as PCR,hybridization, and the like can be used to identify such sequences basedon their sequence homology to the sequence set forth herein. Sequencesisolated based on their sequence identity to the entire ODP2 sequenceset forth herein or to fragments thereof are encompassed by the presentinvention. Such sequences include sequences that are orthologs of thedisclosed sequences. By “orthologs” is intended genes derived from acommon ancestral gene and which are found in different species as aresult of speciation. Genes found in different species are consideredorthologs when their nucleotide sequences and/or their encoded proteinsequences share substantial identity as defined elsewhere herein.Functions of orthologs are often highly conserved among species. Thus,isolated sequences that encode for an ODP2 protein and which hybridizeunder stringent conditions to the ODP2 sequence disclosed herein, or tofragments thereof, are encompassed by the present invention.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any plant of interest. Methods for designingPCR primers and PCR cloning are generally known in the art and aredisclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides, and may belabeled with a detectable group such as ³²P, or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the ODP2 sequences of theinvention. Methods for preparation of probes for hybridization and forconstruction of cDNA and genomic libraries are generally known in theart and are disclosed in Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.).

For example, the entire ODP2 sequence disclosed herein, or one or moreportions thereof, may be used as a probe capable of specificallyhybridizing to corresponding ODP2 sequences and messenger RNAs. Toachieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique among ODP2 sequences and areoptimally at least about 10 nucleotides in length, and at least about 20nucleotides in length. Such probes may be used to amplify correspondingODP2 sequences from a chosen plant by PCR. This technique may be used toisolate additional coding sequences from a desired plant or as adiagnostic assay to determine the presence of coding sequences in aplant. Hybridization techniques include hybridization screening ofplated DNA libraries (either plaques or colonies; see, for example,Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.,Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,optimally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.The duration of the wash time will be at least a length of timesufficient to reach equilibrium.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≥90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is optimal to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.).

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used. Seehttp://www.ncbi.nlm.nih.gov. Alignment may also be performed manually byinspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%sequence identity, optimally at least 80%, more optimally at least 90%,and most optimally at least 95%, compared to a reference sequence usingone of the alignment programs described using standard parameters. Oneof skill in the art will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning, and thelike. Substantial identity of amino acid sequences for these purposesnormally means sequence identity of at least 60%, more optimally atleast 70%, 80%, 90%, and most optimally at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. However, stringent conditions encompasstemperatures in the range of about 1° C. to about 20° C. lower than theT_(m), depending upon the desired degree of stringency as otherwisequalified herein. Nucleic acids that do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides they encode are substantially identical. This may occur,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is when thepolypeptide encoded by the first nucleic acid is immunologically crossreactive with the polypeptide encoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptideindicates that a peptide comprises a sequence with at least 70% sequenceidentity to a reference sequence, optimally 80%, more optimally 85%,most optimally at least 90% or 95% sequence identity to the referencesequence over a specified comparison window. Optimally, optimalalignment is conducted using the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443-453. An indication thattwo peptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution. Peptides that are “substantially similar” share sequencesas noted above except that residue positions that are not identical maydiffer by conservative amino acid changes.

The invention further provides plants, plant cells, and plant partshaving altered levels and/or activities of the ODP2 polypeptides of theinvention. In some embodiments, the plants of the invention have stablyincorporated the ODP2 sequences of the invention. As discussed elsewhereherein, altering the level/activity of the ODP2 sequences of theinvention can produce a variety to phenotypes. As used herein, the termplant includes plant cells, plant protoplasts, plant cell tissuecultures from which plants can be regenerated, plant calli, plantclumps, and plant cells that are intact in plants or parts of plantssuch as embryos, pollen, ovules, seeds, leaves, flowers, branches,fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers,grain and the like. As used herein “grain” is intended the mature seedproduced by commercial growers for purposes other than growing orreproducing the species. Progeny, variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced nucleic acidsequences.

A “subject plant or plant cell” is one in which an alteration, such astransformation or introduction of a polypeptide, has occurred, or is aplant or plant cell which is descended from a plant or cell so alteredand which comprises the alteration. A “control” or “control plant” or“control plant cell” provides a reference point for measuring changes inphenotype of the subject plant or plant cell.

A control plant or plant cell may comprise, for example: (a) a wild-typeplant or cell, i.e., of the same genotype as the starting material forthe alteration which resulted in the subject plant or cell; (b) a plantor plant cell of the same genotype as the starting material but whichhas been transformed with a null construct (i.e. with a construct whichhas no known effect on the trait of interest, such as a constructcomprising a marker gene); (c) a plant or plant cell which is anon-transformed segregant among progeny of a subject plant or plantcell; (d) a plant or plant cell genetically identical to the subjectplant or plant cell but which is not exposed to conditions or stimulithat would induce expression of the gene of interest; or (e) the subjectplant or plant cell itself, under conditions in which the gene ofinterest is not expressed.

METHODS

I. Providing Sequences

The use of the term “nucleotide constructs” or “polynucleotide” hereinis not intended to limit the present invention to nucleotide constructscomprising DNA. Those of ordinary skill in the art will recognize thatnucleotide constructs, particularly polynucleotides andoligonucleotides, comprised of ribonucleotides and combinations ofribonucleotides and deoxyribonucleotides may also be employed in themethods disclosed herein. Thus, the nucleotide constructs of the presentinvention encompass all nucleotide constructs that can be employed inthe methods of the present invention for transforming plants including,but not limited to, those comprised of deoxyribonucleotides,ribonucleotides, and combinations thereof. Such deoxyribonucleotides andribonucleotides include both naturally occurring molecules and syntheticanalogues. The nucleotide constructs of the invention also encompass allforms of nucleotide constructs including, but not limited to,single-stranded forms, double-stranded forms, hairpins, stem-and-loopstructures, and the like.

The nucleic acid sequences of the present invention can beintroduced/expressed in a host cell such as bacteria, yeast, insect,mammalian, or optimally plant cells. It is expected that those of skillin the art are knowledgeable in the numerous systems available for theintroduction of a polypeptide or a nucleotide sequence of the presentinvention. No attempt to describe in detail the various methods knownfor providing proteins in prokaryotes or eukaryotes will be made.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous structural gene isfrom a species different from that from which the structural gene wasderived, or, if from the same species, one or both are substantiallymodified from their original form and/or genomic location.

By “host cell” is meant a cell, which comprises a heterologous nucleicacid sequence of the invention. Host cells may be prokaryotic cells suchas E. coli, or eukaryotic cells such as yeast, insect, amphibian, ormammalian cells. Optimally, host cells are monocotyledonous ordicotyledonous plant cells. A particularly optimal monocotyledonous hostcell is a maize host cell.

The ODP2 sequences of the invention can be provided in expressioncassettes for expression in the plant of interest. The cassette caninclude 5′ and 3′ regulatory sequences operably linked to an ODP2sequence of the invention. “Operably linked” is intended to mean afunctional linkage between two or more elements. For example, anoperable linkage between a polynucleotide of interest and a regulatorysequence (i.e., a promoter) is functional link that allows forexpression of the polynucleotide of interest. Operably linked elementsmay be contiguous or non-contiguous. When used to refer to the joiningof two protein coding regions, by operably linked is intended that thecoding regions are in the same reading frame. The cassette mayadditionally contain at least one additional gene to be cotransformedinto the organism. Alternatively, the additional gene(s) can be providedon multiple expression cassettes. Such an expression cassette isprovided with a plurality of restriction sites for insertion of the ODP2sequence to be under the transcriptional regulation of the regulatoryregions. The expression cassette may additionally contain selectablemarker genes.

The expression cassette can include in the 5′-3′ direction oftranscription, a transcriptional initiation region (i.e., a promoter)and translational initiation region, an ODP2 sequence of the invention,and a transcriptional and translational termination region (i.e.,termination region) functional in plants. The promoter may benative/analogous or foreign to the plant host and/or to the ODP2sequence of the invention. In one embodiment, the promoter employed inthe methods of the invention is the native ODP2 promoter. See, forexample, U.S. Provisional Application No. 60/541,171, entitled “ODP2Promoter and Methods of Use”, filed on Feb. 2, 2004. Additionally, thepromoter may be a natural sequence or alternatively a syntheticsequence. Where the promoter is “foreign” to the plant host, it isintended that the promoter is not found in the native plant into whichthe promoter is introduced. Where the promoter is “foreign” to the ODP2sequence of the invention, it is intended that the promoter is not thenative or naturally occurring promoter for the operably linked ODP2sequence of the invention. As used herein, a chimeric gene comprises acoding sequence operably linked to a transcription initiation regionthat is heterologous to the coding sequence.

While it may be optimal to express the sequences using foreignpromoters, the native promoter sequences may be used. Such constructswould change expression levels of ODP2 in the plant or plant cell. Thus,the phenotype of the plant or plant cell can be altered.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked ODP2 sequence ofinterest, may be native with the plant host, or may be derived fromanother source (i.e., foreign to the promoter, the ODP2 sequence ofinterest, the plant host, or any combination thereof). Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, the gene(s) may be optimized for increased expressionin the transformed plant. That is, the genes can be synthesized usingplant-preferred codons for improved expression. See, for example,Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion ofhost-preferred codon usage. Methods are available in the art forsynthesizing plant-preferred genes. See, for example, U.S. Pat. Nos.5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res.17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, forexample, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus), and humanimmunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991)Nature 353:90-94); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) inMolecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); andmaize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991)Virology 81:382-385). See also, Della-Cioppa et al. (1987) PlantPhysiol. 84:965-968.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues. Marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds,such as glufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). See generally, Yarranton (1992)Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl.Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff(1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon,pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989)Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc.Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg;Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow etal. (1990) Mol. Cell. Biol. 10:3343-3356;

Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Bairnet al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski et al.(1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman (1989) TopicsMol. Struc. Biol. 10:143-162; Degenkolb et al. (1991) Antimicrob. AgentsChemother. 35:1591-1595; Kleinschnidt et al. (1988) Biochemistry27:1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg;Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva etal. (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka et al.(1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag,Berlin); Gill et al. (1988) Nature 334:721-724. Such disclosures areherein incorporated by reference. The above list of selectable markergenes is not meant to be limiting. Any selectable marker gene can beused in the present invention.

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. That is, thenucleic acid can be combined with constitutive, tissue-preferred,developmentally regulated, or other promoters for expression in plants.Constitutive promoters include, for example, the core promoter of theRsyn7 promoter and other constitutive promoters disclosed in WO 99/43838and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al.(1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689);pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten etal. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026),and the like. Other constitutive promoters include, for example, U.S.Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced ODP2expression within a particular plant tissue. Tissue-preferred promotersinclude Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al.(1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. GenGenet. 254(3):337-343; Russell et al. (1997) Transgenic Res.6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341;Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al.(1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant CellPhysiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ.20:181-196; Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138;Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; andGuevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters canbe modified, if necessary, for weak expression.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); and, milps(myo-inositol-1-phosphate synthase); (see WO 00/11177 and U.S. Pat. No.6,225,529; herein incorporated by reference). Gamma-zein is anotherendosperm-specific promoter (Boronat et al. (1986) Plant Science47:95-102). Globulin-1 (Glob-1) is a preferred embryo-specific promoter.For dicots, seed-specific promoters include, but are not limited to,bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, andthe like. For monocots, seed-specific promoters include, but are notlimited to, maize 15 kDa, 22 kDa zein, 27 kDa zein, gamma-zein, waxy,shrunken 1, shrunken 2, globulin 1, etc. See also WO 00/12733, whereseed-preferred promoters from end1 and end2 genes are disclosed; hereinincorporated by reference. Additional seed-preferred promoters includethe oleosin promoter (WO 00/0028058), the lipid transfer protein (LTP)promoter (U.S. Pat. No. 5,525,716). Additional seed-preferred promotersinclude the Lec1 promoter, the Jip1 promoter, and the milps3 promoter(see, WO 02/42424).

The methods of the invention involve introducing a nucleotide constructor a polypeptide into a plant. By “introducing” is intended presentingto the plant the nucleotide construct (i.e., DNA or RNA) or apolypeptide in such a manner that the nucleic acid or the polypeptidegains access to the interior of a cell of the plant. The methods of theinvention do not depend on a particular method for introducing thenucleotide construct or the polypeptide to a plant, only that thenucleotide construct gains access to the interior of at least one cellof the plant. Methods for introducing nucleotide constructs and/orpolypeptide into plants are known in the art including, but not limitedto, stable transformation methods, transient transformation methods, andvirus-mediated methods.

By “stable transformation” is intended that the nucleotide constructintroduced into a plant integrates into the genome of the plant and iscapable of being inherited by progeny thereof. By “transienttransformation” is intended that a nucleotide construct or thepolypeptide introduced into a plant does not integrate into the genomeof the plant.

Thus the ODP2 sequences of the invention can be provided to a plantusing a variety of transient transformation methods including, but notlimited to, the introduction of ODP2 protein or variants thereofdirectly into the plant and the introduction of the an ODP2 transcriptinto the plant. Such methods include, for example, microinjection orparticle bombardment. See, for example, Crossway et al. (1986) Mol Gen.Genet. 202:179-185; Nomura et al. (1986) Plant Sci. 44:53-58; Hepler etal. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994)The Journal of Cell Science 107:775-784, all of which are hereinincorporated by reference. Alternatively, the various viral vectorsystems can be used for transient expression or the ODP2 nucleotideconstruct can be precipitated in a manner that precludes subsequentrelease of the DNA (thus, transcription from the particle-bound DNA canoccur, but the frequency with which its released to become integratedinto the genome is greatly reduced). Such methods include the use ofPEI, as outlined in more detail in Example 13.

The nucleotide constructs of the invention may be introduced into plantsby contacting plants with a virus or viral nucleic acids. Generally,such methods involve incorporating a nucleotide construct of theinvention within a viral DNA or RNA molecule. It is recognized that thean ODP2 polypeptide of the invention may be initially synthesized aspart of a viral polyprotein, which later may be processed by proteolysisin vivo or in vitro to produce the desired recombinant protein. Further,it is recognized that promoters of the invention also encompasspromoters utilized for transcription by viral RNA polymerases. Methodsfor introducing nucleotide constructs into plants and expressing aprotein encoded therein, involving viral DNA or RNA molecules, are knownin the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190,5,866,785, 5,589,367 and 5,316,931; herein incorporated by reference.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No.5,563,055; Zhao et al., U.S. Pat. No. 5,981,840), direct gene transfer(Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particleacceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050;Tomes et al., U.S. Pat. No. 5,879,918; Tomes et al., U.S. Pat. No.5,886,244; Bidney et al., U.S. Pat. No. 5,932,782; Tomes et al. (1995)“Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment,” in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe etal. (1988) Biotechnology 6:923-926); and Lec1 transformation (WO00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet.22:421-477; Sanford et al. (1987) Particulate Science and Technology5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674(soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean);Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182(soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean);Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988)Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988)Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buisinget al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995)“Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment,” in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al.(1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984)Nature (London) 311:763-764; Bowen et al., U.S. Pat. No. 5,736,369(cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,the polynucleotide of the invention can be contained in transfercassette flanked by two non-recombinogenic recombination sites. Thetransfer cassette is introduced into a plant having stably incorporatedinto its genome a target site which is flanked by two non-recombinogenicrecombination sites that correspond to the sites of the transfercassette. An appropriate recombinase is provided and the transfercassette is integrated at the target site. The polynucleotide ofinterest is thereby integrated at a specific chromosomal position in theplant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplant species of interest include, but are not limited to, corn (Zeamays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularlythose Brassica species useful as sources of seed oil, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). Optimally, plants of the present inventionare crop plants (for example, corn, alfalfa, sunflower, Brassica,soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco,etc.), more optimally corn and soybean plants, yet more optimally cornplants.

Plants of particular interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

Typically, an intermediate host cell will be used in the practice ofthis invention to increase the copy number of the cloning vector. Withan increased copy number, the vector containing the nucleic acid ofinterest can be isolated in significant quantities for introduction intothe desired plant cells. In one embodiment, plant promoters that do notcause expression of the polypeptide in bacteria are employed.

Prokaryotes most frequently are represented by various strains of E.coli; however, other microbial strains may also be used. Commonly usedprokaryotic control sequences which are defined herein to includepromoters for transcription initiation, optionally with an operator,along with ribosome binding sequences, include such commonly usedpromoters as the beta lactamase (penicillinase) and lactose (lac)promoter systems (Chang et al. (1977) Nature 198:1056), the tryptophan(trp) promoter system (Goeddel et al. (1980) Nucleic Acids Res. 8:4057)and the lambda derived P L promoter and N-gene ribosome binding site(Shimatake et al. (1981) Nature 292:128). The inclusion of selectionmarkers in DNA vectors transfected in E coli. is also useful. Examplesof such markers include genes specifying resistance to ampicillin,tetracycline, or chloramphenicol.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva et al. (1983) Gene22:229-235); Mosbach et al. (1983) Nature 302:543-545).

A variety of eukaryotic expression systems such as yeast, insect celllines, plant and mammalian cells, are known to those of skill in theart. As explained briefly below, a polynucleotide of the presentinvention can be expressed in these eukaryotic systems. In someembodiments, transformed/transfected plant cells, as discussed infra,are employed as expression systems for production of the proteins of theinstant invention.

Synthesis of heterologous polynucleotides in yeast is well known(Sherman et al. (1982) Methods in Yeast Genetics, Cold Spring HarborLaboratory). Two widely utilized yeasts for production of eukaryoticproteins are Saccharomyces cerevisiae and Pichia pastoris. Vectors,strains, and protocols for expression in Saccharomyces and Pichia areknown in the art and available from commercial suppliers (e.g.,Invitrogen). Suitable vectors usually have expression control sequences,such as promoters, including 3-phosphoglycerate kinase or alcoholoxidase, and an origin of replication, termination sequences and thelike as desired.

A protein of the present invention, once expressed, can be isolated fromyeast by lysing the cells and applying standard protein isolationtechniques to the lists. The monitoring of the purification process canbe accomplished by using Western blot techniques or radioimmunoassay ofother standard immunoassay techniques.

The sequences of the present invention can also be ligated to variousexpression vectors for use in transfecting cell cultures of, forinstance, mammalian, insect, or plant origin. Illustrative cell culturesuseful for the production of the peptides are mammalian cells. A numberof suitable host cell lines capable of expressing intact proteins havebeen developed in the art, and include the HEK293, BHK21, and CHO celllines. Expression vectors for these cells can include expression controlsequences, such as an origin of replication, a promoter (e.g. the CMVpromoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter),an enhancer (Queen et al. (1986) Immunol. Rev. 89:49), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites (e.g., an SV40 large T Ag poly A additionsite), and transcriptional terminator sequences. Other animal cellsuseful for production of proteins of the present invention areavailable, for instance, from the American Type Culture Collection.

Appropriate vectors for expressing proteins of the present invention ininsect cells are usually derived from the SF9 baculovirus. Suitableinsect cell lines include mosquito larvae, silkworm, armyworm, moth andDrosophila cell lines such as a Schneider cell line (See, Schneider(1987) J. Embryol. Exp. Morphol. 27:353-365).

As with yeast, when higher animal or plant host cells are employed,polyadenylation or transcription terminator sequences are typicallyincorporated into the vector. An example of a terminator sequence is thepolyadenylation sequence from the bovine growth hormone gene. Sequencesfor accurate splicing of the transcript may also be included. An exampleof a splicing sequence is the VP1 intron from SV40 (Sprague et al.(1983) J. Virol. 45:773-781). Additionally, gene sequences to controlreplication in the host cell may be incorporated into the vector such asthose found in bovine papilloma virus type-vectors (Saveria-Campo (1985)DNA Cloning Vol. II a Practical Approach, D. M. Glover, Ed., IRL Press,Arlington, Va., pp. 213-238).

Animal and lower eukaryotic (e.g., yeast) host cells are competent orrendered competent for transfection by various means. There are severalwell-known methods of introducing DNA into animal cells. These include:calcium phosphate precipitation, fusion of the recipient cells withbacterial protoplasts containing the DNA, treatment of the recipientcells with liposomes containing the DNA, DEAE dextrin, electroporation,biolistics, and micro-injection of the DNA directly into the cells. Thetransfected cells are cultured by means well known in the art (Kuchler(1997) Biochemical Methods in Cell Culture and Virology, Dowden,Hutchinson and Ross, Inc.).

In some embodiments, the content and/or composition of polypeptides ofthe present invention in a plant may be modulated by altering, in vivoor in vitro, the promoter of a gene to up- or down-regulate geneexpression. In some embodiments, the coding regions of native genes ofthe present invention can be altered via substitution, addition,insertion, or deletion to decrease activity of the encoded enzyme. See,e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868. Inother embodiments, the polypeptide of the invention is introduced. Andin some embodiments, an isolated nucleic acid (e.g., a vector)comprising a promoter sequence is transfected into a plant cell.Subsequently, a plant cell comprising the promoter operably linked to apolynucleotide of the present invention is selected for by means knownto those of skill in the art such as, but not limited to, Southern blot,DNA sequencing, or PCR analysis using primers specific to the promoterand to the gene and detecting amplicons produced therefrom. A plant orplant part altered or modified by the foregoing embodiments is grownunder plant forming conditions for a time sufficient to modulate theconcentration and/or composition of polypeptides of the presentinvention in the plant. Plant forming conditions are well known in theart and discussed briefly, supra.

A method for modulating the concentration and/or activity of thepolypeptide of the present invention is provided. By “modulation” isintended any alteration in the level and/or activity (i.e., increase ordecrease) that is statistically significant compared to a control plantor plant part. In general, concentration, composition or activity isincreased or decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% relative to a control plant, plant part, or cell. Themodulation may occur during and/or subsequent to growth of the plant tothe desired stage of development. Modulating nucleic acid expressiontemporally and/or in particular tissues can be controlled by employingthe appropriate promoter operably linked to a polynucleotide of thepresent invention in, for example, sense or antisense orientation asdiscussed in greater detail, supra. Induction of expression of apolynucleotide of the present invention can also be controlled byexogenous administration of an effective amount of inducing compound.Inducible promoters and inducing compounds, which activate expressionfrom these promoters, are well known in the art. In specificembodiments, the polypeptides of the present invention are modulated inmonocots, particularly maize.

The level of the ODP2 polypeptide may be measured directly, for example,by assaying for the level of the ODP2 polypeptide in the plant, orindirectly, for example, by measuring the ODP2 activity of the ODP2polypeptide in the plant. Methods for determining the presence of ODP2activity are described elsewhere herein.

In specific embodiments, the polypeptide or the polynucleotide of theinvention is introduced into the plant cell. Subsequently, a plant cellhaving the introduced sequence of the invention is selected usingmethods known to those of skill in the art such as, but not limited to,Southern blot analysis, DNA sequencing, PCR analysis, or phenotypicanalysis. A plant or plant part altered or modified by the foregoingembodiments is grown under plant forming conditions for a timesufficient to modulate the concentration and/or activity of polypeptidesof the present invention in the plant. Plant forming conditions are wellknown in the art and discussed briefly elsewhere herein.

It is also recognized that the level and/or activity of the polypeptidemay be modulated by employing a polynucleotide that is not capable ofdirecting, in a transformed plant, the expression of a protein or anRNA. For example, the polynucleotides of the invention may be used todesign polynucleotide constructs that can be employed in methods foraltering or mutating a genomic nucleotide sequence in an organism. Suchpolynucleotide constructs include, but are not limited to, RNA:DNAvectors, RNA:DNA mutational vectors, RNA:DNA repair vectors,mixed-duplex oligonucleotides, self-complementary RNA:DNAoligonucleotides, and recombinogenic oligonucleobases. Such nucleotideconstructs and methods of use are known in the art. See, U.S. Pat. Nos.5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972; and 5,871,984;all of which are herein incorporated by reference. See also, WO98/49350, WO 99/07865, WO 99/25821, and Beetham et al. (1999) Proc.Natl. Acad. Sci. USA 96:8774-8778; herein incorporated by reference.

It is therefore recognized that methods of the present invention do notdepend on the incorporation of the entire polynucleotide into thegenome, only that the plant or cell thereof is altered as a result ofthe introduction of the polynucleotide into a cell. In one embodiment ofthe invention, the genome may be altered following the introduction ofthe polynucleotide into a cell. For example, the polynucleotide, or anypart thereof, may incorporate into the genome of the plant. Alterationsto the genome of the present invention include, but are not limited to,additions, deletions, and substitutions of nucleotides into the genome.While the methods of the present invention do not depend on additions,deletions, and substitutions of any particular number of nucleotides, itis recognized that such additions, deletions, or substitutions comprisesat least one nucleotide.

In some embodiments, the activity and/or level of the ODP2 polypeptideof the invention is increased. An increase in the level or activity ofthe ODP2 polypeptide of the invention can be achieved by providing tothe plant an ODP2 polypeptide. As discussed elsewhere herein, manymethods are known the art for providing a polypeptide to a plantincluding, but not limited to, direct introduction of the polypeptideinto the plant and/or introducing into the plant (transiently or stably)a nucleotide construct encoding a polypeptide having ODP2 activity. Inother embodiments, the level or activity of an ODP2 polypeptide may beincreased by altering the gene encoding the ODP2 polypeptide or itspromoter. See, e.g. U.S. Pat. No. 5,565,350 and PCT/US93/03868. Theinvention therefore encompasses mutagenized plants that carry mutationsin ODP2 genes, where the mutations increase expression of the ODP2 geneor increase the ODP2 activity of the encoded ODP2 polypeptide.

In some embodiments, the activity and/or level of the ODP2 polypeptideof the invention of is reduced or eliminated by introducing into a planta polynucleotide that inhibits the level or activity of the ODP2polypeptide of the invention. The polynucleotide may inhibit theexpression of ODP2 directly, by preventing translation of the ODP2messenger RNA, or indirectly, by encoding a polypeptide that inhibitsthe transcription or translation of an ODP2 gene encoding an ODP2protein. Methods for inhibiting or eliminating the expression of a genein a plant are well known in the art, and any such method may be used inthe present invention to inhibit the expression of ODP2 in a plant. Inother embodiments of the invention, the activity of ODP2 polypeptide isreduced or eliminated by transforming a plant cell with an expressioncassette comprising a polynucleotide encoding a polypeptide thatinhibits the activity of the ODP2 polypeptide. In other embodiments, theactivity of an ODP2 polypeptide may be reduced or eliminated bydisrupting the gene encoding the ODP2 polypeptide. The inventionencompasses mutagenized plants that carry mutations in ODP2 genes, wherethe mutations reduce expression of the ODP2 gene or inhibit the ODP2activity of the encoded ODP2 polypeptide.

Reduction of the activity of specific genes (also known as genesilencing or gene suppression) is desirable for several aspects ofgenetic engineering in plants. Methods for inhibiting gene expressionare well known in the art and include, but are not limited to,homology-dependent gene silencing, antisense technology, RNAinterference (RNAi), and the like. The general term homology-dependentgene silencing encompasses the phenomenon of cis-inactivation,trans-inactivation, and cosuppression. See Finnegan et al. (1994)Biotech. 12:883-888; and Matzke et al. (1995) Plant Physiol.107:679-685; both incorporated herein in their entirety by reference.These mechanisms represent cases of gene silencing that involvetransgene/transgene or transgene/endogenous gene interactions that leadto reduced expression of protein in plants. A “transgene” is arecombinant DNA construct that has been introduced into the genome by atransformation procedure. As one alternative, incorporation of antisenseRNA into plants can be used to inhibit the expression of endogenousgenes and produce a functional mutation within the genome. The effect isachieved by introducing into the cell(s) DNA that encodes RNA that iscomplementary to the sequence of mRNA of the target gene. See e.g. Birdet al. (1991) Biotech and Gen. Eng. Rev. 9:207-226; incorporated hereinin its entirety by reference. See also the more detailed discussionherein below addressing these and other methodologies for achievinginhibition of expression or function of a gene.

Many techniques for gene silencing are well known to one of skill in theart, including, but not limited to, antisense technology (see, e.g.,Sheehy et al. (1988) Proc. Natl. Acad. Sci. USA 85:8805-8809; and U.S.Pat. Nos. 5,107,065; 5,453,566; and 5,759,829); cosuppression (e.g.,Taylor (1997) Plant Cell 9:1245; Jorgensen (1990) Trends Biotech.8(12):340-344; Flavell (1994) Proc. Natl. Acad. Sci. USA 91:3490-3496;Finnegan et al. (1994) Bio/Technology 12:883-888; and Neuhuber et al.(1994) Mol. Gen. Genet. 244:230-241); RNA interference (Napoli et al.(1990) Plant Cell 2:279-289; U.S. Pat. No. 5,034,323; Sharp (1999) GenesDev. 13:139-141; Zamore et al. (2000) Cell 101:25-33; and Montgomery etal. (1998) Proc. Natl. Acad. Sci. USA 95:15502-15507), virus-inducedgene silencing (Burton et al. (2000) Plant Cell 12:691-705; andBaulcombe (1999) Curr. Op. Plant Bio. 2:109-113); target-RNA-specificribozymes (Haseloff et al. (1988) Nature 334: 585-591); hairpinstructures (Smith et al. (2000) Nature 407:319-320; WO 99/53050; WO02/00904; WO 98/53083; Chuang and Meyerowitz (2000) Proc. Natl. Acad.Sci. USA 97:4985-4990; Stoutjesdijk et al. (2002) Plant Physiol.129:1723-1731; Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4:29-38;Pandolfini et al. BMC Biotechnology 3:7, U.S. Patent Publication No.20030175965; Panstruga et al. (2003) Mol. Biol. Rep. 30:135-140; Wesleyet al. (2001) Plant J. 27:581-590; Wang and Waterhouse (2001) Curr.Opin. Plant Biol. 5:146-150; U.S. Patent Publication No. 20030180945;and, WO 02/00904, all of which are herein incorporated by reference);ribozymes (Steinecke et al. (1992) EMBO J. 11:1525; and Perriman et al.(1993) Antisense Res. Dev. 3:253); oligonucleotide-mediated targetedmodification (e.g., WO 03/076574 and WO 99/25853); Zn-finger targetedmolecules (e.g., WO 01/52620; WO 03/048345; and WO 00/42219); transposontagging (Maes et al. (1999) Trends Plant Sci. 4:90-96; Dharmapuri andSonti (1999) FEMS Microbiol. Lett. 179:53-59; Meissner et al. (2000)Plant J. 22:265-274; Phogat et al. (2000) J. Biosci. 25:57-63; Walbot(2000) Curr. Opin. Plant Biol. 2:103-107; Gal et al. (2000) NucleicAcids Res. 28:94-96; Fitzmaurice et al. (1999) Genetics 153:1919-1928;Bensen et al. (1995) Plant Cell 7:75-84; Mena et al. (1996) Science274:1537-1540; and U.S. Pat. No. 5,962,764); each of which is hereinincorporated by reference; and other methods or combinations of theabove methods known to those of skill in the art.

It is recognized that with the polynucleotides of the invention,antisense constructions, complementary to at least a portion of themessenger RNA (mRNA) for the ODP2 sequences can be constructed.Antisense nucleotides are constructed to hybridize with thecorresponding mRNA. Modifications of the antisense sequences may be madeas long as the sequences hybridize to and interfere with expression ofthe corresponding mRNA. In this manner, antisense constructions having70%, optimally 80%, more optimally 85% sequence identity to thecorresponding antisensed sequences may be used. Furthermore, portions ofthe antisense nucleotides may be used to disrupt the expression of thetarget gene. Generally, sequences of at least 50 nucleotides, 100nucleotides, 200 nucleotides, 300, 400, 450, 500, 550, or greater may beused.

The polynucleotides of the present invention may also be used in thesense orientation to suppress the expression of endogenous genes inplants. Methods for suppressing gene expression in plants usingpolynucleotides in the sense orientation are known in the art. Themethods generally involve transforming plants with a DNA constructcomprising a promoter that drives expression in a plant operably linkedto at least a portion of a polynucleotide that corresponds to thetranscript of the endogenous gene. Typically, such a nucleotide sequencehas substantial sequence identity to the sequence of the transcript ofthe endogenous gene, optimally greater than about 65% sequence identity,more optimally greater than about 85% sequence identity, most optimallygreater than about 95% sequence identity. See, U.S. Pat. Nos. 5,283,184and 5,034,323; herein incorporated by reference. Thus, many methods maybe used to reduce or eliminate the activity of an ODP2 polypeptide. Morethan one method may be used to reduce the activity of a single ODP2polypeptide. In addition, combinations of methods may be employed toreduce or eliminate the activity of the ODP2 polypeptides.

Furthermore, it is recognized that the methods of the invention mayemploy a nucleotide construct that is capable of directing, in atransformed plant, the expression of at least one protein, or at leastone RNA, such as, for example, an antisense RNA that is complementary toat least a portion of an mRNA. Typically such a nucleotide construct iscomprised of a coding sequence for a protein or an RNA operably linkedto 5′ and 3′ transcriptional regulatory regions. Alternatively, it isalso recognized that the methods of the invention may employ anucleotide construct that is not capable of directing, in a transformedplant, the expression of a protein or an RNA.

The ODP2 polynucleotides of the present invention can also be combinedwith genes implicated in transcriptional regulation, homeotic generegulation, stem cell maintenance and proliferation, cell division,and/or cell differentiation such as other ODP2 homologues; Wuschel (see,e.g, Mayer et al. (1998) Cell 95:805-815); clavata (e.g., CLV1, CVL2,CLV3) (see, e.g., WO 03/093450; Clark et al. (1997) Cell 89:575-585;Jeong et al. (1999) Plant Cell 11:1925-1934; Fletcher et al. (1999)Science 283:1911-1914); Clavata and Embryo Surround region genes (e.g.,CLE) (see, e.g., Sharma et al. (2003) Plant Mol. Biol. 51:415-425; Hobeet al. (2003) Dev Genes Evol 213:371-381; Cock & McCormick (2001) PlantPhysiol 126:939-942; and Casamitjana-Martinez et al. (2003) Curr Biol13:1435-1441); baby boom (e.g., BNM3, BBM) (see, e.g., WO 00/75530;Boutiler et al. (2002) Plant Cell 14:1737-1749); Zwille (Lynn et al.(1999) Dev 126:469-481); leafy cotyledon (e.g., Lec1, Lec2) (see, e.g.,Lotan et al. (1998) Cell 93:1195-1205; WO 00/28058; Stone et al. (2001)PNAS 98:11806-11811; and U.S. Pat. No. 6,492,577); Shoot Meristem-less(STM) (Long et al. (1996) Nature 379:66-69); ultrapetala (ULT) (see,e.g., Fletcher (2001) Dev 128:1323-1333); mitogen activated proteinkinase (MAPK) (see, e.g., Jonak et al. (2002) Curr Opin Plant Biol5:415); kinase associated protein phosphatase (KAPP) (see, e.g.,Williams et al. (1997) PNAS 94:10467-10472; and Trotochaud et al. (1999)Plant Cell 11:393-406); ROP GTPase (see, e.g., Wu et al. (2001) PlantCell 13:2841-2856; and Trotochaud et al. (1999) Plant Cell 11:393-406);fasciata (e.g., FAS1, FAS2) (see, e.g., Kaya et al. (2001) Cell104:131-142); cell cycle genes (see, e.g., U.S. Pat. No. 6,518,487; WO99/61619; and WO 02/074909), Shepherd (SHD) (see, e.g., Ishiguro et al.(2002) EMBO J. 21:898-908); Poltergeist (see, e.g., Yu et al. (2000) Dev127:1661-1670; Yu et al. (2003) Curr Biol 13:179-188); Pickle (PKL)(see, e.g., Ogas et al. (1999) PNAS 96:13839-13844); knox genes (e.g.,KN1, KNAT1) (see, e.g., Jackson et al. (1994) Dev 120:405-413; Lincolnet al. (1994) Plant Cell 6:1859-1876; Venglat et al. (2002) PNAS99:4730-4735); fertilization independent endosperm (FIE) (e.g., Ohad etal. (1999) Plant Cell 11:407-415), and the like, the disclosures ofwhich are herein incorporated by reference. The combinations generatedcan also include multiple copies of any one of the polynucleotides ofinterest. The combinations may have any combination of up-regulating anddown-regulating expression of the combined polynucleotides. Thecombinations may or may not be combined on one construct fortransformation of the host cell, and therefore may be providedsequentially or simultaneously. The host cell may be a wild-type ormutant cell, in a normal or aneuploid state.

II. Altering the Oil Content in Plants

The present invention provides a method for altering the oil content ofa plant. By “altering the oil phenotype” of a plant is intended anymodulation (increase or decrease) in the overall level of oil in theplant or plant part (i.e., seed) when compared to a control plant. Thealtered oil phenotype can comprise any statistically significantincrease or decrease in oil when compared to a control plant. Forexample, altering the oil phenotype can comprise either an increase or adecrease in overall oil content of about 0.1%, 0.5%, 1%, 3% 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or greater when compared to a control plant or plant partthat has not be transformed with the ODP2 sequence of the invention.Alternatively, the alteration in oil phenotype can include about a 0.5fold, 1 fold, 2 fold, 4 fold, 8 fold, 16 fold, or 32 fold increase inoverall oil phenotype in the plant or plant part when compared to acontrol plant that has not been transformed with the ODP2 sequence.

It is further recognized that the alteration in the oil phenotype neednot be an overall increase/decrease in oil content, but also includes achange in the ratio of various components of the plant oil (i.e., achange in the ratio of any of the various fatty acids that compose theplant oil). For example, the ratio of various fatty acids such aslinoleic acid, oleic acid, palmitic acid, stearic acid, myristic acid,linolenic acid, lauric acid, and the like, could be altered and therebychange the oil phenotype of the plant or plant part when compared to acontrol plant lacking the ODP2 sequence of the invention.

The method for altering the oil phenotype of a plant comprises providingan ODP2 sequence of the invention. An ODP2 polypeptide can be providedby introducing the polypeptide into the plant, and thereby modifying theoil content of the plant or plant part. Alternatively, an OPD2nucleotide sequence can be provided by introducing into the plant aheterologous polynucleotide comprising an ODP2 nucleotide sequence ofthe invention, expressing the ODP2 sequence, and thereby modifying theoil content of the plant. In yet other embodiments, the ODP2 nucleotideconstruct introduced into the plant is stably incorporated into thegenome of the plant.

Methods for determining if the oil phenotype of the plant has beenaltered are known in the art. For example, the oil phenotype can bedetermined using NMR. Briefly, data for plant or plant part oilpercentage, total plant or plant part oil, and plant or plant partweight are collected and analyzed by NMR. If changes from the control (aplant not transformed with ODP2) are observed above base-line, a PCRco-segregation analysis can be performed to determine if the changes arecorrelated with the presence of the ODP2 sequence. In specificembodiments, the plant part is an embryo. Alternatively, fatty acidcontent and composition can be determined by gas chromatography (GC).See, for example, WO 03/001902, herein incorporated by reference.

As discussed above, one of skill will recognize the appropriate promoterto use to alter the oil content of the plant in the desired manner.Exemplary promoters for this embodiment include the ubiquitin promoter(Christensen et al. (1992) Plant Molecular Biology 18:675-680), a lipidtransfer protein (LTP) promoter (U.S. Pat. No. 5,525,716), a gamma-zeinpromoter (GZP) (Boronat et al. (1986) Plant Sciences 47:95-102), and theoleosin promoter (WO 00/28058), the lec1 promoter (WO 02/42424), and theZm-ODP2 promoter (U.S. Provisional Application No. 60/541,171, entitled“ODP2 Promoter and Methods of Use” filed on Feb. 2, 2004, hereinincorporated by reference in its entirety.

In specific embodiments, the oil content of the plant is decreased uponincreasing level/activity of the ODP2 polypeptide in a plant. Adecreased oil content finds use in the wet milling industry and in theethanol dry grind industry. In the dry grind process, raw corn isground, mixed with water, cooked, saccharified, fermented, and thendistilled to make ethanol. The process also recovers distillers driedgains with solubles that can be used in feed products. Various methodsof ethanol dry grind are known in the art. See, for example, U.S. Pat.Nos. 6,592,921, 6,433,146, Taylor et al. (2003) Applied Biochemistry andBiotechnology 104:141-148; Taylor et al. (2000) Biotechnol Prog.16:541-7, and Taylor et al. (2001) Appl Biocehm Biotechnol 94:41-9.

In the wet milling process, the purpose is to fractionate the kernel andisolate chemical constituents of economic value into their componentparts. The process allows for the fractionation of starch into a highlypurified form, as well as, for the isolation in crude forms of othermaterial including, for example, unrefined oil, or as a wide mix ofmaterials which commonly receive little to no additional processingbeyond drying. Hence, in the wet milling process grain is softened bysteeping and cracked by grinding to release the germ from the kernels.The germ is separated from the heavier density mixture of starch, hullsand fiber by “floating” the germ segments free of the other substancesin a centrifugation process. This allows a clean separation of theoil-bearing fraction of the grain from tissue fragments that contain thebulk of the starch. Since it is not economical to extract oil on a smallscale, many wet milling plants ship their germ to large, centralized oilproduction facilities. Oil is expelled or extracted with solvents fromdried germs and the remaining germ meal is commonly mixed into corngluten feed (CGF), a coproduct of wet milling. Hence, starch containedwithin the germ is not recovered as such in the wet milling process andis channeled to CGF. See, for example, Anderson et al. (1982) “The CornMilling Industry”; CRC Handbook of Processing and Utilization inAgriculture, A. Wolff, Boca Raton, Fla., CRC Press., Inc., Vol. 11, Part1, Plant Products: 31-61 and Eckhoff (Jun. 24-26, 1992) Proceedings ofthe 4th Corn Utilization Conference, St. Louis, Mo., printed by theNational Corn Growers Association, CIBA-GEIGY Seed Division, and theUSDA, both of which are herein incorporated by reference.

In other embodiments, the oil content of the plant or plant part isincreased. Plants containing an increase in oil content can be used in avariety of applications. For example, high oil plants have an improvedfood efficiency, which results in greater amounts of energy in the germ.In addition, high oil plants can have an increase in lysine levels,reduced dust during grinding, and improved feed product when comparedwith normal plants. High oil content in seeds also yields greateramounts of oil when grain is processed into oil and provides economicadvantages to starch wet milling.

Accordingly, the present invention further provides plants having analtered oil phenotype when compared to the oil phenotype of a controlplant. In specific embodiments, the altered oil phenotype is in a grain.In some embodiments, the plant of the invention has an increasedlevel/activity of the ODP2 polypeptide of the invention and has adecreased oil content. In other embodiments, such plants have stablyincorporated into their genome a heterologous nucleic acid moleculecomprising an ODP2 nucleotide sequence of the invention operably linkedto a promoter that drives expression in the plant cell.

III. Altering Starch Production in Plants

The present invention provides a method for modifying the starchproduction of a plant. By “starch” is intended a polymer of glucose andnormally comprises amylose, amylopectin or a mixture of these twopolymer types. Functionally analogous chemical compounds, also includedwithin the definition of starch, include phytoglycogen (which occurs inselect types of corn) and water soluble polysaccharides (glucosepolymers lacking the crystalline structure of starch granules).

By “modify starch production” of a plant is intended any modulation(increase or decrease) in the overall level of starch in the plant orplant part (i.e., seed, grain, etc.) when compared to a control plant.The modification in starch production can comprise any statisticallysignificant increase or decrease in starch levels when compared to acontrol plant. For example, modifying starch production can compriseeither an increase or a decrease in overall starch content of about0.1%, 0.5%, 1%, 3% 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 110%, 125% or greater whencompared to a control plant or plant part that has not be transformedwith the ODP2 sequence of the invention. Alternatively, the modificationof starch production can include about a 0.2 fold, 0.5 fold, 1 fold, 2fold, 4 fold, 8 fold, 16 fold, or 32 fold increase in overall starchcontent in the plant or plant part when compared to a control plant thathas not been transformed with the ODP2 sequence.

The method for modifying the starch production in a plant comprisesproviding an ODP2 sequence of the invention. An ODP2 polypeptide can beprovided by introducing the polypeptide into the plant, and therebymodifying the starch production of the plant or plant part.Alternatively, an ODP2 nucleotide sequence can be provided byintroducing into the plant a heterologous polynucleotide comprising anODP2 nucleotide sequence of the invention, expressing the ODP2 sequence,and thereby modifying the starch production of the plant. In yet otherembodiments, the ODP2 nucleotide construct introduced into the plant isstably incorporated into the genome of the plant.

Methods for determining if the starch production in the plant or plantpart has been altered are known in the art. For example, total starchmeasurement can be performed as outlined in McCleary et al. (1994)Journal of Cereal Science 20:51-58, McCleary et al. (1997) J. Assoc.Off. Anal. Chem 80:571-579, and McCleary et al. (2002) J. AOACInternational 85:1103-1111, each of which is herein incorporated byreference.

As discussed above, one of skill will recognize the appropriate promoterto use to modify starch production in a plant in the desired manner.Exemplary promoters for this embodiment include the ubiquitin promoter(Christensen et al. (1992) Plant Molecular Biology 18:675-680), a lipidtransfer protein (LTP) promoter (U.S. Pat. No. 5,525,716), a gamma-zeinpromoter (GZP) (Boronat et al. (1986) Plant Sciences 47:95-102), and theoleosin promoter (WO 00/28058), the lec1 promoter (WO 02/42424), and theZm-ODP2 promoter (U.S. Provisional Application No. 60/541,171, entitled“ODP2 Promoter and Methods of Use” filed on Feb. 2, 2004.

In specific embodiments, the modification of starch production resultsin an increase in starch content in the plant or plant part uponincreasing level/activity of the ODP2 polypeptide in a plant. Anincreased starch content finds use in the in the wet milling industryand in the ethanol dry grind industry. In other embodiments, the starchproduction results in a decrease in starch content in the plant or plantpart upon decreasing the level/activity of the ODP2 polypeptide in theplant.

Accordingly, the present invention further provides plants or plantparts having modified starch production when compared to the starchproduction of a control plant or plant part. In specific embodiments,the plant having the altered starch production is a grain. In someembodiments, the plant of the invention has an increased level/activityof the ODP2 polypeptide of the invention and has an increase in starchaccumulation. In other embodiments, such plants have stably incorporatedinto their genome a heterologous nucleic acid molecule comprising anODP2 nucleotide sequence of the invention operably linked to a promoterthat drives expression in the plant cell.

IV. Modifying the Regenerative Capacity of Plants

The present invention further provides methods to modify theregenerative capacity of a plant. As used herein “regeneration” refersto a morphogenic response that results in the production of new tissues,organs, embryos, whole plants or parts of whole plants that are derivedfrom a single cell or a group of cells. Regeneration may proceedindirectly via a callus phase or directly, without an intervening callusphase. “Regenerative capacity” refers to the ability of a plant cell toundergo regeneration.

In this embodiment, the method of modifying the regenerative capacity ofa plant comprises providing an ODP2 sequence of the invention. In oneembodiment, the regenerative capcity of the plant is modified byincreasing the level and/or activity of an ODP2 polypeptide. The ODP2sequence can be provided by introducing an ODP2 polypeptide into theplant, and thereby modifying the regenerative capacity of said plant.Alternatively, an ODP2 nucleotide sequence can be provided byintroducing into the plant a heterologous polynucleotide comprising anODP2 polynucleotide of the invention, expressing the ODP2 sequence, andthereby modifying the regenerative capacity of the plant. In yet otherembodiments, the ODP2 nucleotide construct introduced into the plant isstably incorporated into the genome of the plant.

It is further recognized that providing the ODP2 sequences may be usedto enhance the regenerative capacity of plant tissues both in vitro andin vivo and thereby stimulating cell proliferation and/ordifferentiation. In one embodiment, a method of initiating meristemformation is provided.

As discussed in further detail below, the promoter used to express theODP2 sequence of the invention will depend, in part, on the targettissue used for regeneration. Various promoters of interest includeconstitutive promoters, tissue-preferred promoters, developmentallyregulated promoters, and chemically-inducible systems. Various promotersthat regulate ovule and embryo expression, nucellus expression, andinner integument expression are discussed in further detail below.

The ODP2 sequences of the invention also will be useful for inducingapomixis in plants. In specific embodiments, increasing the level and/oractivity of the ODP2 polypeptide induces apomixis. Apomixis and methodsof conferring apomixis into plants are discussed in U.S. Pat. Nos.5,710,367; 5,811,636; 6,028,185; 6,229,064; and 6,239,327 as well as WO00/24914, all of which are incorporated herein by reference.Reproduction in plants is ordinarily classified as sexual or asexual.The term apomixis is generally accepted as the replacement of sexualreproduction by various forms of asexual reproduction (Rieger et al.(1976) Glossary of Genetics and Cytogenetics, Springer-Verlag, New York,N.Y.). In general, the initiation of cell proliferation in the embryoand endosperm are uncoupled from fertilization. Apomixis is agenetically controlled method of reproduction in plants where the embryois formed without the union of an egg and a sperm. There are three basictypes of apomictic reproduction: 1) apospory-embryo develops from achromosomally unreduced egg in an embryo sac derived from a somatic cellin the nucellus; 2) diplospory-embryo develops from an unreduced egg inan embryo sac derived from the megaspore mother cell; and, 3)adventitious embryony-embryo develops directly from a somatic cell. Inmost forms of apomixis, pseudogamy or fertilization of the polar nucleito produce endosperm is necessary for seed viability.

These types of apomixis have economic potential because they can causeany genotype, regardless of how heterozygous, to breed true. It is areproductive process that bypasses female meiosis and syngamy to produceembryos genetically identical to the maternal parent. With apomicticreproduction, progeny of specially adaptive or hybrid genotypes wouldmaintain their genetic fidelity throughout repeated life cycles. Inaddition to fixing hybrid vigor, apomixis can make possible commercialhybrid production in crops where efficient male sterility or fertilityrestoration systems for producing hybrids are not known or developed.Apomixis can make hybrid development more efficient. It also simplifieshybrid production and increases genetic diversity in plant species withgood male sterility. It also provides a system for the production ofhybrid seed in species, or between genotypes of the same species inwhich crossing between separate parent plants is impractical on a largescale.

In another embodiment, methods for producing embryogenic cells areprovided. By “embryogenic cell” is intended a cell that has completedthe transition from either a somatic or a gametophytic cell to a statewhere no further applied stimuli are necessary to produce an embryo. Inthis embodiment, the method comprises providing an ODP2 sequence of theinvention. In one embodiment, the level and/or activity of the ODP2polypeptide is increased and thereby allows for an increased productionof embryogenic cells. In one embodiment, the ODP2 sequence is an ODP2polypeptide which is provided by introducing the polypeptide into theplant, and thereby producing an embryogenic cell. Alternatively, an OPD2nucleotide sequence can be provided by introducing into the plant aheterologous polynucleotide comprising an ODP2 nucleotide sequence ofthe invention, expressing the ODP2 sequence, and thereby producing anembryogenic cell. In yet other embodiments, the ODP2 nucleotideconstruct introduced into the plant is stably incorporated into thegenome of the plant.

Further provided is a method for producing asexually derived embryos. Asused herein, the term “asexually derived embryo” refers to an embryothat is generated in the absence of fertilization. The term is inclusiveof apomitic and somatic embryos. The term “somatic embryogenesis” refersto non-zygotic embryogenesis. The method comprises introducing into aplant an ODP2 sequence of the invention and thereby producing asexuallyderived embryos. As discussed above, the embryo can be a somatic embryo,an adventitious embryos, or a gametophytic embryo.

Methods are also provided for an increase in the production of somaticembryos in a plant. In one embodiment, the level and/or activity of theODP2 polypeptide is increased and thereby allowing for the production ofsomatic embryos. In one embodiment, an ODP2 sequence of the invention isprovided. The polypeptide can be provided by introducing the polypeptideinto the plant, and thereby increasing the production of somaticembryos. Alternatively, an OPD2 nucleotide sequence can be provided byintroducing into the plant a heterologous polynucleotide comprising anODP2 nucleotide sequence of the invention, expressing the ODP2 sequence,and thereby increasing the production of somatic embryos. In yet otherembodiments, the ODP2 nucleotide construct introduced into the plant isstably incorporated into the genome of the plant.

The somatic embryo structures may form as individual embryos or as acluster of structures. In specific embodiments, the plants (i.e., theroot, leaf, seedling) expressing the ODP2 sequences are cultured invitro. The embryos, non-embryogenic callus or both are transferred toappropriate media for the production of embryos or plantlets. While thesomatic embryo can be formed independent of additional growthregulators, it is recognized that in some embodiments, growth regulatorscan be added to the media and include, but are not limited to, 2,4-D(Mordhorst et al. (1998) Genetics 149:549-563).

An increase in asexually derived embryos can be assayed by determiningif embryogenesis or embryonic callus is initiated at a higher frequencyfrom transgenic lines expressing ODP2 sequences of the inventioncompared to a control plant or plant part. See, for example, Boutiler etal. (2002) The Plant Cell 14:1737-1749, herein incorporated byreference.

It is recognized that the plant having the somatic embryo structures mayform only a limited number of somatic embryo structures and then resumeadditional post germination growth. In other embodiments, expression ofthe ODP2 sequence leads to the reiteration of the embryo formingprocess, with the result that new embryos or cotyledons are formedcontinuously.

In particular embodiments, the level and/or activity of the ODP2polypeptide will be reduced prior to the regeneration of a plant fromthese various embryogenic cell types. Methods for reducing the activityof the ODP2 polypeptide are discussed in detail elsewhere herein.

Embryogenesis can be induced in haploid cells, such as pollen cells, eggcells, or cells from haploid lines, to produce haploid plants. Methodsof inducing embryogenesis in haploid cells comprise providing an ODP2sequence of the invention to a plant. In one embodiment, the leveland/or activity of the ODP2 polypeptide is increased and thereby allowsfor the induction of embryogenesis in haploid cells. An ODP2 polypeptidecan be provided by introducing the polypeptide into the plant, andthereby inducing embryogenesis. Alternatively, an OPD2 nucleotidesequence can be provided by introducing into the plant a heterologouspolynucleotide comprising an ODP2 nucleotide sequence of the invention,expressing the ODP2 sequence, and thereby inducing embryogenesis. In yetother embodiments, the ODP2 nucleotide construct introduced into theplant is stably incorporated into the genome of the plant.

In one embodiment, the ODP2 nucleotide sequence introduced into theplant is under the control of a tissue specific promoter that is activein a haploid cell or tissue or a promoter that is active duringmicrospore development (such as, the maize PG47 promoter (Allen et al.(1993) Plant J. 3:261-71), the zm-G13 promoter (Hamilton et al. (1992)Plant Mol Biol. 18:211-218). In other embodiments, the ODP2 nucleotidesequence is under the control of an inducible promoter and theapplication of the inducer allows expression of the ODP2 sequencetherein. Alternatively, the promoter used can be both inducible andtissue-preferred, giving greater control over the process. For example,the promoter can be both haploid-tissue specific and inducible. In oneembodiment, the promoter is an inducible pollen-specific promoter usedto induce somatic embryogenesis in pollen cells. In still otherembodiments, site-specific recombination systems can be used incombination with promoters (i.e., constitutive promoters or induciblepromoters) to regulate the appropriate time and level of ODP2expression. Thus, the methods of the invention find use in promotingembryogenesis in microspore and anther cultures.

Providing the ODP2 sequence to a haploid tissue or cell results in theformation of haploid somatic embryos, which can be grown into haploidplants using standard techniques. When an inducible promoter is used(whether tissue specific or not), an optimal method comprises exposingexcised transgenic tissue containing the haploid cells (e.g., pollen orovules) to the inducer specific for the inducible promoter for a timesufficient to induce the formation of a somatic embryo, withdrawing theinducer, and growing the somatic embryo into a transgenic haploid plantin the absence of the inducer.

Diploidization of the haploid plants to form dihaploids, eitherspontaneously or by treatment with the appropriate chemical (e.g.colchicine) will significantly expedite the process of obtaininghomozygous plants as compared to a method of conventional geneticsegregation. This technology will not only be beneficial for breedingpurposes but also for basic research such as studies of mutagenesis andother genetic studies, because dihaploids are truly homozygous down tothe DNA level, containing two identical copies of each gene.

In yet another embodiment, adventitious embryony can be achieved byproviding an ODP2 sequence of the invention to sporophytic ovule tissuessuch as the nucellus, the inner integuments, or other tissues lyingadjacent to or in proximity to the developing embryo sac.

The ODP2 sequences of the invention may also be used as a selectablemarker to recover transgenic plants. In one embodiment, the level and/oractivity of the ODP2 sequence is increased. In this embodiment, a plantis transformed with the ODP2 sequences along with a nucleotide sequenceof interest. Upon expression of the ODP2 sequences, the plants can beselected based on their ability to regenerate under conditions in whichwild type explants are unable to. For example, the transgenic plants maybe able to regenerate in the absence of growth regulators. If the ODP2sequence and the polynucleotide of interest are carried on separateplasmids, the ODP2 sequence can be subsequently removed from transgenicplants by routine breeding methods.

One of skill in the art will recognize that a variety of promoters canbe used in the various methods of the invention. Somatic or gametophyticembryos can be obtained expressing the ODP2 polypeptide under thecontrol of constitutive promoters, tissue-preferred, developmentallyregulated, or various inducible promoters including chemical inductionsystems (i.e., tetracycline-inducible systems, steroid induciblepromoters, and ethanol-inducible promoters). Temporal and/or spatialrestriction of ODP2 is optimal when recurrent embryogenesis is not adesirable trait. Promoters of interest when microspore-derived embryoproduction is desired include, but are not limited to, microspore/pollenexpressed genes such as NTM19 (EP 790,311), BCP1 (Xu et al. (1995) PlantMol. Biol. 22:573-588, PG47 (Allen et al. (1993) Plant J. 3:261-71),ZmG13 (Hamilton et al. (1992) Plant Mol. Biol. 18:211-218), and BNM1(Treacy et al. (1997) Plant Mol. Biol. 34:603-611), each reference isherein incorporated by reference. Promoters of interest when theproduction of somatic embryos are desired include, but are not limitedto, cytokinin inducible IB6 and CK11 promoters (Brandstatter et al.(1998) Plant Cell 10:1009-1019). Exemplary promoters of interest whenadventitious embryony, diplospory or haploid parthenogenesis of embryosac components, include, the AtDMC1 gene (WO 98/28431), promoters thatdirect expression in the ovule, such as the AGL11 promoter (Rounsley etal. (1995) Plant Cell 10:1009-1019) and the SERK promoter (Schmidt etal. (1997) Development 124:2049-2062), promoters that direct expressionin the nucellus such as the NUC1 promoter (WO 98/08961), promoters thatregulate expression of inner integument genes such as the FBP7 promoter(Angenent et al. (1995) Plant Cell 7:1569-1582),microspore/pollen-preferred promoters (discussed above) and chemicalinduction systems. Each of these references is herein incorporated byreference.

Accordingly, the present invention further provides plants having amodified regenerative capacity, including plants that are capable ofproducing asexually derived embryos. In some embodiments, the plantshaving a modified regenerative capacity have an increased level/activityof the ODP2 polypeptide of the invention. In other embodiments, theplant comprises a heterologous ODP2 nucleotide sequence of the inventionoperably linked to a promoter that drives expression in the plant cell.In other embodiments, such plants have stably incorporated into theirgenome a heterologous nucleic acid molecule comprising an ODP2nucleotide sequence of the invention operably linked to a promoter thatdrives expression in the plant cell.

In other embodiments, the OPD2 sequences of the invention can be used tomodify the tolerance of a plant to abiotic stress. In one embodiment, amethod is provided to increase or maintain seed set during abioticstress episodes. During periods of stress (i.e., drought, salt, heavymetals, temperature, etc.) embryo development is often aborted. Inmaize, halted embryo development results in aborted kernels on the ear.Preventing this kernel loss will maintain yield. Accordingly, methodsare provided to increase the stress resistance in a plant (i.e., anearly developing embryo).

The method comprises providing an ODP2 sequence of the invention. Thepolypeptide can be provided by introducing the polypeptide into theplant, and thereby modifying the plants tolerance to abiotic stress.Alternatively, an OPD2 nucleotide sequence can be provided byintroducing into the plant a heterologous polynucleotide comprising anODP2 nucleotide sequence of the invention, expressing the ODP2 sequence,and thereby modifying the plants tolerance to abiotic stress. In yetother embodiments, the ODP2 nucleotide construct introduced into theplant is stably incorporated into the genome of the plant.

A variety of promoters can be employed in this method. In oneembodiment, the ODP2 sequence is under the control of an early promoter.An early embryo is defined as the stages of embryo development includingthe zygote and the developing embryo up to the point where embryomaturation begins. An “early embryo promoter” is a promoter that drivesexpression predominately during the early stages of embryo development(i.e., before 15-18 DAP). Alternatively, the early embryo promoter candrive expression during both early and late stages. Early embryopromoters include, but are not limited to, to Lec 1 (WO 02/42424); cim1,a pollen and whole kernel specific promoter (WO 00/11177); theseed-preferred promoter end1 (WO 00/12733); and, the seed-preferredpromoter end2 (WO 00/12733) and 1pt2 (U.S. Pat. No. 5,525,716).Additional promoter include, smi1ps, an embryo specific promoter andcz19B1 a whole kernel specific promoter. See, for example, WO 00/11177,which is herein incorporated by reference. All of these references isherein incorporated by reference.

Methods to assay for an increase in seed set during abiotic stress areknown in the art. For example, plants having the ODP2 sequences of theinvention can be monitored under various stress conditions and comparedto controls plants (not having had the ODP2 introduced). For instance,the plant having the OPD2 sequence can be subjected to various degreesof stress during flowering and seed set. Under identical conditions, thegenetically modified plant having the ODP2 sequences will have a highernumber of developing kernels than a wild type (non-transformed) plant.

Accordingly, the present invention further provides plants havingincreased yield or maintaining their yield during periods of abioticstress (i.e. drought, salt, heavy metals, temperature, etc). In someembodiments, the plants having an increased or maintained yield duringabiotic stress have an increased level/activity of the ODP2 polypeptideof the invention. In other embodiments, the plant comprises aheterologous ODP2 nucleotide sequence of the invention operably linkedto a promoter that drives expression in the plant cell. In otherembodiments, such plants have stably incorporated into their genome aheterologous nucleic acid molecule comprising an ODP2 nucleotidesequence of the invention operably linked to a promoter that drivesexpression in the plant cell.

V. Modifying the Transformation Efficiency in Plants

The present invention provides novel methods for transformation and forincreasing transformation frequencies. As used herein “responsive targetplant cell” is a plant cell that exhibits increased transformationefficiency after the introduction of the ODP2 sequences of the inventionwhen compared to a control plant or plant part. The increase intransformation efficiency can comprise any statistically significantincrease when compared to a control plant. For example, an increase intransformation efficiency can comprises about 0.2%, 0.5%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 120%, 125% or greater increase when compared to a controlplant or plant part. Alternatively, the increase in transformationefficiency can include about a 0.2 fold, 0.5 fold, 1 fold, 2 fold, 4fold, 8 fold, 16 fold, or 32 fold or greater increase in transformationefficiency in the plant when compared to a control plant or plant part.

Many maize genotypes, and in particular elite germplasm developed incommercial breeding programs, are recalcitrant to in vitro culture andtransformation. Such genotypes do not produce an appropriate embryogenicor organogenic culture response on culture media developed to elicitsuch responses from typically suitable explants such as immatureembryos. Furthermore, when exogenous DNA is introduced into theseimmature embryos (for example, using particle bombardment orAgrobacterium), no transgenic events are recovered after selection (orso few events are recovered as to make transformation of such a genotypeimpractical). When the ODP2 gene is expressed (either transiently orstably) in immature embryos of such genotypes, vigorously growingtransgenic events can be readily recovered.

Thus, the present invention finds use in increasing the transformationof a recalcitrant plant or explants. As used herein “recalcitrant plantor explant” means a plant or explant that is more difficult to transformthan model systems. In maize such a model system is High type-II maize.Elite maize inbreds are typically recalcitrant. In soybeans such modelsystems are Peking or Jack.

In one embodiment of the invention, a method for increasing thetransformation efficiency in a plant is provided. The method comprisesproviding an ODP2 sequence of the invention. An ODP2 polypeptide can beprovided by introducing the polypeptide into the plant, and therebyincreasing the transformation efficiency of the plant. Alternatively, anOPD2 nucleotide sequence can be provided by introducing into the plant aheterologous polynucleotide comprising an ODP2 nucleotide sequence ofthe invention, expressing the ODP2 sequence, and thereby increasing thetransformation efficiency of the plant. In yet other embodiments, theODP2 nucleotide construct introduced into the plant is stablyincorporated into the genome of the plant. Through the introduction ofan ODP2 into a recalcitrant plant and producing a positive influence ontransformation, the methods of the invention provide the potential toincrease the overall genetic transformation throughput of variousrecalcitrant germplasm.

Accordingly, the present invention further provides plants havingincreased transformation efficiencies when compared to thetransformation efficiency of a control plant. In some embodiments, theplants having increased transformation efficiencies have an increasedlevel/activity of the ODP2 polypeptide of the invention. In otherembodiments, the plant comprises a heterologous ODP2 nucleotide sequenceof the invention operably linked to a promoter that drives expression inthe plant cell. In other embodiments, such plants have stablyincorporated into their genome a heterologous nucleic acid moleculecomprising an ODP2 nucleotide sequence of the invention operably linkedto a promoter that drives expression in the plant cell.

In another embodiment, a method of transforming in a plant is provided.The method comprises providing a target plant, where the target planthad been provided an ODP2 sequence of the invention. In someembodiments, the OPD2 nucleotide sequence is provided by introducinginto the plant a heterologous polynucleotide comprising an ODP2nucleotide sequence of the invention, expressing the ODP2 sequence. Inyet other embodiments, the ODP2 nucleotide construct introduced into thetarget plant is stably incorporated into the genome of the plant. Thetarget plant is transformed with a polynucleotide of interest. It isrecognized that the target plant having had the ODP2 sequence introduced(referred to herein as a “modified target plant”), can be grown underconditions to produce at least one cell division to produce a progenycell expressing the ODP2 sequence prior to transformation with one ormore polynucleotides of interest. As used herein “re-transformation”refers to the transformation of a modified cell.

The modified target cells having been provided the ODP2 sequence can beobtained from T0 transgenic cultures, regenerated plants or progenywhether grown in vivo or in vitro so long as they exhibit stimulatedgrowth compared to a corresponding cell that does not contain themodification. This includes but is not limited to transformed callus,tissue culture, regenerated T0 plants or plant parts such as immatureembryos or any subsequent progeny of T0 regenerated plants or plantparts.

Once the target cell is provided with the ODP2 nucleotide sequence it isre-transformed with at least one gene of interest. The transformed cellcan be from transformed callus, transformed embryo, T0 regeneratedplants or its parts, progeny of T0 plants or parts thereof as long asthe ODP2 sequence of the invention is stably incorporated into thegenome.

Methods to determine transformation efficiencies or the successfultransformation of the polynucleotide of interest are known in the art.For example, transgenic plants expressing a selectable marker can bescreened for transmission of the gene(s) of interest using, for example,chemical selection, phenotype screening standard immunoblot and DNAdetection techniques. Transgenic lines are also typically evaluated onlevels of expression of the heterologous nucleic acid. Expression at theRNA level can be determined initially to identify and quantitateexpression-positive plants. Standard techniques for RNA analysis can beemployed and include PCR amplification assays using oligonucleotideprimers designed to amplify only the heterologous RNA templates andsolution hybridization assays using heterologous nucleic acid-specificprobes.

The RNA-positive plants can then be analyzed for protein expression byWestern immunoblot analysis using the specifically reactive antibodiesof the present invention. In addition, in situ hybridization andimmunocytochemistry according to standard protocols can be done usingheterologous nucleic acid specific polynucleotide probes and antibodies,respectively, to localize sites of expression within transgenic tissue.Generally, a number of transgenic lines are usually screened for theincorporated nucleic acid to identify and select plants with the mostappropriate expression profiles.

Seeds derived from plants regenerated from re-transformed plant cells,plant parts or plant tissues, or progeny derived from the regeneratedplants, may be used directly as feed or food, or further processing mayoccur.

Any polynucleotide of interest can be used in the methods of theinvention. Various changes in phenotype are of interest includingmodifying the fatty acid composition in a plant, altering the amino acidcontent, starch content, or carbohydrate content of a plant, altering aplant's pathogen defense mechanism, affecting kernel size, sucroseloading, and the like. The gene of interest may also be involved inregulating the influx of nutrients, and in regulating expression ofphytate genes particularly to lower phytate levels in the seed. Theseresults can be achieved by providing expression of heterologous productsor increased expression of endogenous products in plants. Alternatively,the results can be achieved by providing for a reduction of expressionof one or more endogenous products, particularly enzymes or cofactors inthe plant. These changes result in a change in phenotype of thetransformed plant.

Genes of interest are reflective of the commercial markets and interestsof those involved in the development of the crop. Crops and markets ofinterest change, and as developing nations open up world markets, newcrops and technologies will emerge also. In addition, as ourunderstanding of agronomic traits and characteristics such as yield andheterosis increase, the choice of genes for transformation will changeaccordingly. General categories of genes of interest include, forexample, those genes involved in information, such as zinc fingers,those involved in communication, such as kinases, and those involved inhousekeeping, such as heat shock proteins. More specific categories oftransgenes, for example, include genes encoding important traits foragronomics, insect resistance, disease resistance, herbicide resistance,sterility, grain characteristics, and commercial products. Genes ofinterest include, generally, those involved in oil, starch,carbohydrate, or nutrient metabolism as well as those affecting kernelsize, sucrose loading, and the like.

Agronomically important traits such as oil, starch, and protein contentcan be genetically altered in addition to using traditional breedingmethods. Modifications include increasing content of oleic acid,saturated and unsaturated oils, increasing levels of lysine and sulfur,providing essential amino acids, and also modification of starch.Hordothionin protein modifications are described in U.S. Pat. Nos.5,703,049, 5,885,801, 5,885,802, and 5,990,389, herein incorporated byreference. Another example is lysine and/or sulfur rich seed proteinencoded by the soybean 2S albumin described in U.S. Pat. No. 5,850,016,and the chymotrypsin inhibitor from barley, described in Williamson etal. (1987) Eur. J. Biochem. 165:99-106, the disclosures of which areherein incorporated by reference.

Derivatives of the coding sequences can be made by site-directedmutagenesis to increase the level of preselected amino acids in theencoded polypeptide. For example, methionine-rich plant proteins such asfrom sunflower seed (Lilley et al. (1989) Proceedings of the WorldCongress on Vegetable Protein Utilization in Human Foods and AnimalFeedstuffs, ed. Applewhite (American Oil Chemists Society, Champaign,Ill.), pp. 497-502; herein incorporated by reference); corn (Pedersen etal. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359;both of which are herein incorporated by reference); and rice (Musumuraet al. (1989) Plant Mol. Biol. 12:123, herein incorporated by reference)could be used. Other agronomically important genes encode latex, Floury2, growth factors, seed storage factors, and transcription factors.

Insect resistance genes may encode resistance to pests that have greatyield drag such as rootworm, cutworm, European Corn Borer, and the like.Such genes include, for example, Bacillus thuringiensis toxic proteingenes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756;5,593,881; and Geiser et al. (1986) Gene 48:109); and, the like.

Genes encoding disease resistance traits include detoxification genes,such as against fumonosin (U.S. Pat. No. 5,792,931); avirulence (avr)and disease resistance (R) genes (Jones et al. (1994) Science 266:789;Martin et al. (1993) Science 262:1432; and Mindrinos et al. (1994) Cell78:1089); and the like.

Herbicide resistance traits may include genes coding for resistance toherbicides that act to inhibit the action of acetolactate synthase(ALS), in particular the sulfonylurea-type herbicides (e.g., theacetolactate synthase (ALS) gene containing mutations leading to suchresistance, in particular the S4 and/or Hra mutations), genes coding forresistance to herbicides that act to inhibit action of glutaminesynthase, such as phosphinothricin or basta (e.g., the bar gene),glyphosate (e.g., the EPSPS gene and the GAT gene; see, for example,U.S. Publication No. 20040082770 and WO 03/092360) or other such genesknown in the art. The bar gene encodes resistance to the herbicidebasta, the nptII gene encodes resistance to the antibiotics kanamycinand geneticin, and the ALS-gene mutants encode resistance to theherbicide chlorsulfuron.

Sterility genes can also be encoded in an expression cassette andprovide an alternative to physical detasseling. Examples of genes usedin such ways include male tissue-preferred genes and genes with malesterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210.Other genes include kinases and those encoding compounds toxic to eithermale or female gametophytic development.

The quality of grain is reflected in traits such as levels and types ofoils, saturated and unsaturated, quality and quantity of essential aminoacids, and levels of cellulose. In corn, modified hordothionin proteinsare described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and5,990,389.

Commercial traits can also be encoded on a gene or genes that couldincrease for example, starch for ethanol production, or provideexpression of proteins. Another important commercial use of transformedplants is the production of polymers and bioplastics such as describedin U.S. Pat. No. 5,602,321. Genes such as β-Ketothiolase, PHBase(polyhydroxyburyrate synthase), and acetoacetyl-CoA reductase (seeSchubert et al. (1988) J. Bacteriol. 170:5837-5847) facilitateexpression of polyhyroxyalkanoates (PHAs).

Exogenous products include plant enzymes and products as well as thosefrom other sources including prokaryotes and other eukaryotes. Suchproducts include enzymes, cofactors, hormones, and the like. The levelof proteins, particularly modified proteins having improved amino aciddistribution to improve the nutrient value of the plant, can beincreased. This is achieved by the expression of such proteins havingenhanced amino acid content.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Cloning of ZM-ODP2

The protein encoded by maize EST clone cpflc.pk009.f4 was initiallyidentified as the homologue of a rice putative ovule development protein(BAB89946). The EST clone was subjected to full-insert sequencing.Comparison of rice BAB89946 and the protein sequence encoded by thelongest open reading frame (ORF) from cpflc.pk009.f4 suggests that thisclone may have an internal deletion which causes premature terminationof the protein by at least 120 amino acids. A genomic fragmentencompassing the potential deletion was amplified by PCR using DNAisolated from Hi II callus. Sequencing results confirm the presence ofan extra 146 base pairs in the genomic fragment. When added to cDNAclone cpflc.pk009.f4, this 146-bp can be read through in the samereading frame and the ORF is extended to encode a protein very similarto BAB89946 in length.

The full-length Zm-ODP2 (SEQ ID NO:1) used in the transformation wascreated by combining the 5′ end of cDNA clone cpflc.pk009.f4 and part ofthe genomic clone from Hi II callus that contains the missing 146-bp.More specifically, a 1790-bp EcoRI-SbfI fragment from cpflc.pk009.f4 anda 582-bp SbfI-SalI genomic fragment were ligated into pBluescript II KS+digested with EcoRI and SalI to form PHP20430.

The full-length Zm-ODP2 sequence is 2260 nucleotides in length. The openreading frame is 2133 nucleotides in length and starts at nucleotide 128and ends at nucleotide 2260 of SEQ ID NO:1. The nucleotide sequence ofthe Zm-ODP2 open reading frame is set forth in SEQ ID NO:3. The 710amino acid sequence encoded by the Zm-ODP2 sequence is set forth in SEQID NO:2.

Example 2 Sequence Analysis of Zm-ODP2

The ZM-ODP2 sequence of the invention was analyzed for conserveddomains. FIG. 1 shows an alignment of the amino acid sequence of Zm-ODP2(SEQ ID NO:2) with various polypeptides sharing sequence similarity tothe Zm-ODP2 sequence. Specifically, Zm-ODP2 shares over its full-lengthabout 65.4% sequence identity and 72.7% sequence similarity with OsAnt(Accession No. BAB89946; SEQ ID NO:26). Zm-ODP2 shares over itsfull-length about 57.1% sequence identity and about 62.3% sequencesimilarity to OSBNM (Accession No. AAL47205; SEQ ID NO:27). Zm-ODP2shares over its full-length about 42% sequence identity and about 53.2%sequence similarity to OSODP (Accession No. CAE05555; SEQ ID NO:29).Zm-ODP2 shares over its full-length about 37% sequence identity andabout 45% sequence similarity to BnBBM2 (Accession No. AAM33801; SEQ IDNO:33). Zm-ODP2 shares over its full-length about 38% sequence identityand about 47% sequence similarity to BnBBM1 (AAM33800; SEQ ID NO:32).Zm-ODP2 shares over its full-length about 38.1% sequence identity andabout 46.3% sequence similarity to ATBBM (Accession No. AAM33803; SEQ IDNO:31). Zm-ODP2 shares over its full-length about 40% sequence identityand about 43% sequence similarity to AtODP (Accession No. AAD30633; SEQID NO:36). Zm-ODP2 shares over its full-length about 35.6% sequenceidentity and about 50% sequence similarity to ATODP (Accession No.NP_175530; SEQ ID NO:34). Zm-ODP2 shares over its full-length about34.9% sequence identity and about 44.6% sequence similarity to AtODP(Accession No. BAB02492; SEQ ID NO:35). Zm-ODP2 shares over itsfull-length about 38.4% sequence identity and about 46% sequencesimilarity to AtODP (NP_197245; SEQ ID NO:30). A consensus sequence forall 11 aligned polypeptides is also provided (SEQ ID NO:37).

All 11 proteins present in the alignment have two AP2 (APETALA2;pfam00847.8) domains. Using the amino acid numbering of the Zm-ODP2, thefirst AP2 domain is from about amino acid 273 to about 343 and thesecond AP2 domain is from about amino acid 375 to about 437. Theconsensus sequence for the APETALA2 PFAM family isSKYRGVRQRPWGKWVAEIRDPRKGTRVWLGTFDTAEEAARAYDVAALKLRGPSAVLNFPNEL (SEQ IDNO: 38).

Example 3 Variants of Zm-ODP2

A. Variant Nucleotide Sequences of Zm-ODP2 (SEQ ID NO:1) That Do NotAlter the Encoded Amino Acid Sequence

The Zm-ODP2 nucleotide sequence set forth in SEQ ID NO:1 was used togenerate 6 variant nucleotide sequences having the nucleotide sequenceof the open reading frame with about 70.6%, 76.1%, 81.2%, 86.3%, 92.1%,and 97.1% nucleotide sequence identity when compared to the startingunaltered ORF nucleotide sequence of SEQ ID NO:1. These functionalvariants were generated using a standard codon table. While thenucleotide sequence of the variant was altered, the amino acid sequenceencoded by the open reading frame did not change.

The variants of Zm-ODP2 using this method are set forth in SEQ IDNOS:6-11. Specifically, SEQ ID NO: 6 shares about 97.1% nucleic acidsequence identity to the Zm-ODP2 sequence of SEQ ID NO:1; SEQ ID NO: 7shares about 92.1% nucleic acid sequence identity to SEQ ID NO:1, SEQ IDNO:8 shares about 86.3% nucleic acid sequence identity to SEQ ID NO:1;SEQ ID NO:9 shares about 81.2% nucleic acid sequence identity to SEQ IDNO:1; SEQ ID NO:10 shares about 76.1% nucleic acid sequence identity toSEQ ID NO:1; and SEQ ID NO:11 shares about 70.6% nucleic acid sequenceidentity to SEQ ID NO:1.

B. Variant Amino Acid Sequences of Zm-ODP2

Variant amino acid sequences of Zm-ODP2 were generated. In this example,one amino acid was altered. Specifically, the open reading frame setforth in SEQ ID NO:3 was reviewed to determined the appropriate aminoacid alteration. The selection of the amino acid to change was made byconsulting the protein alignment (with the other orthologs and othergene family members from various species). See FIG. 1. An amino acid wasselected that was deemed not to be under high selection pressure (nothighly conserved) and which could be rather easily substituted by anamino acid with similar chemical characteristics (i.e., similarfunctional side-chain). Using the protein alignment set forth in FIG. 1and focusing at the N-terminus (amino acids 1-50), the serine at aminoacid position 37 (shaded) was changed to a threonine, which ischemically similar. Thus, the “TCC” serine codon in the nucleic acidsequence is changed to an “ACC” codon for threonine. The Zm-ODP2sequence having the single change from “TCC” to “ACC” is set forth inSEQ ID NO:12.

Once the targeted amino acid was identified, the procedure outlined inExample 3A was followed. Variants having about 70.4% (SEQ ID NO:18),75.9% (SEQ ID NO:17), 81.5% (SEQ ID NO:16), 86.6% (SEQ ID NO:15), 91.9%(SEQ ID NO:14), and 97.3% (SEQ ID NO:13) nucleic acid sequence identityto SEQ ID NO:3 were generated using this method. SEQ ID NOS: 13-18 allencode the same polypeptide, which is set forth in SEQ ID NO: 19.

C. Additional Variant Amino Acid Sequences of Zen-ODP2

In this example, artificial protein sequences were created at a narrowerinterval range (82.5%, 87.5%, 92.5%, and 97.5% identity relative to thereference protein sequence). This latter effort requires identifyingconserved and variable regions from the alignment set forth in FIG. 1and then the judicious application of an amino acid substitutions table.These parts will be discussed in more detail below.

Largely, the determination of which amino acid sequences were alteredwas made based on the conserved regions among AP2 protein or among theother ODP-like genes. See FIG. 1. Based on the sequence alignment, thevarious regions of the Zm-ODP2 that can likely be altered arerepresented in lower case letters, while the conserved regions arerepresented by capital letters. It is recognized that conservativesubstitutions can be made in the conserved regions below withoutaltering function. In addition, one of skill will understand thatfunctional variants of the ODP2 sequence of the invention can have minornon-conserved amino acid alterations in the conserved domain. Thissequence is set forth in SEQ ID NO:2.

MAtvNNWLAFSLSPqelppsqttdstlisaatADhvsGDVCFNipqdwsmrgselsalvaepkledflggisfseqhhkancnmipstsetvcyassgastgyhhqlyhqptssalhfadsvmvassagvhdggamlsaaaangvagaasanGGGIGLSMIKNWLRSQPapmqprvaaaegaqglslsmnmagttqgaagmpllagerarapesvstsaqggavvvtapkedsggsgvagalvavstdtggsggasadntaRKTVDTFGQRTSIYRGVTRHRWTGRYEAHLWDNSCRREGQTRKGRQVYLGGYDKEEKAARAYDLAALKYWGATTTTNFPVSNYEKELEDMKHMTRQEFVASLRRKSSGFSRGASIYRGVTRHHQHGRWQARIGRVAGNKDLYLGTFSTQEEAAEAYDIAAIKFRGLNAVTNFDMSRYDVKSILDSSALPIGSAAKRLKEAEAaasaqhhhagvvsydvgriasqlgdggalaaaygahyhgaawptiafqpgaastglyhpyagqpmrgggwckqeqdhaviaaahslqdlhhlnlgaagandffsagqqaaaaamhglgsidsaslehSTGSNSVVYNGGvgdsngasavgGSGGGYmmpmsaagatttsamvsheqvharaydeakqaaqmGYESYLVnaenngggrmsawgtvvsaaaaaaassndnmaaDVGHGG AQLFSVWNDTThe conserved regions are found between about amino acid 1-2; 5-14;33-34; 38-43; 153-169; 262-463; 591-602; 614-619; 655-661; and 695-800of SEQ ID NO: 2. The non-conserved regions are from about amino acids3-4; 15-32; 35-37; 44-152; 170-261; 464-590; 603-613; 620-654; and662-694 of SEQ ID NO: 2.

The goal was to create four artificial protein sequences that aredifferent from the original in the intervals of 80-85%, 85-90%, 90-95%,and 95-100% identity. Midpoints of these intervals were targeted, withliberal latitude of plus or minus 1%, for example. The amino acidssubstitutions will be effected by a custom Perl script. The substitutiontable is provided below in Table 1.

TABLE 1 Substitution Table Strongly Similar and Rank of Amino OptimalOrder to Acid Substitution Change Comment I L, V 1 50:50 substitution LI, V 2 50:50 substitution V I, L 3 50:50 substitution A G 4 G A 5 D E 6E D 7 W Y 8 Y W 9 S T 10 T S 11 K R 12 R K 13 N Q 14 Q N 15 F Y 16 M L17 First methionine cannot change H Na No good substitutes C Na No goodsubstitutes P Na No good substitutes

First, any conserved amino acids in the protein that should not bechanged was identified and “marked off” for insulation from thesubstitution. The start methionine will of course be added to this listautomatically. Next, the changes were made.

H, C, and P will not be changed in any circumstance. The changes willoccur with isoleucine first, sweeping N-terminal to C-terminal. Thenleucine, and so on down the list until the desired target it reached.Interim number substitutions can be made so as not to cause reversal ofchanges. The list is ordered 1-17, so start with as many isoleucinechanges as needed before leucine, and so on down to methionine. Clearlymany amino acids will in this manner not need to be changed. L, I and Vwill involved a 50:50 substitution of the two alternate optimalsubstitutions.

Four amino acid sequences were written as output. Perl script was usedto calculate the percent identities. Using this procedure, variants ofZm-ODP2 were generating having about 82.4% (SEQ ID NO:23), 87.3% (SEQ IDNO:22), 92.4% (SEQ ID NO:21), and 97.3% (SEQ ID NO:20) amino acididentity to the starting unaltered ORF nucleotide sequence of SEQ IDNO:2. FIG. 2 provides an amino acid alignment of SEQ ID NO:2 and themodified proteins set forth in SEQ ID NOS: 20-23.

TABLE 2 Summary of the ODP2 sequences and exemplary variants thereof(SEQ ID NOS 1-25) SEQ Nucleotide ID or NO Amino Acid Description ofSequence 1 nucleic acid ZM-ODP2 full length 2 amino acid ZM-ODP2 fulllength 3 nucleic acid ZM-ODP2 - open reading frame 4 nucleic acidZM-ODP2 cDNA insert from EST clone cpf1c.pk009.f4 5 nucleic acid cDNAinsert from EST clone cpc1c.pk005.c19 6 nucleic acid Nucleic acidvariant of Zm-ODP2 having 97.2% nucleic acid sequence identity to SEQ IDNO: 2 7 nucleic acid Nucleic acid variant of Zm-ODP2 having 92.1%nucleic acid sequence identity to SEQ ID NO: 2 8 nucleic acid Nucleicacid variant of Zm-ODP2 having 86.3% nucleic acid sequence identity toSEQ ID NO: 2 9 nucleic acid Nucleic acid variant of Zm-ODP2 having 81.2%nucleic acid sequence identity to SEQ ID NO: 2 10 nucleic acid Nucleicacid variant of Zm-ODP2 having 76.1% nucleic acid sequence identity toSEQ ID NO: 2 11 nucleic acid Nucleic acid variant of Zm-ODP2 having70.6% nucleic acid sequence identity to SEQ ID NO: 2 12 nucleic acidVariant of Zm-ODP2 having the serine 37 codon altered from “tcc” to thethreonine codon of “acc”. The ORF encodes the amino acid sequence setforth in SEQ ID NO: 19. 13 nucleic acid Variant of Zm-ODP2 having 97.3%nucleic acid sequence identity to SEQ ID NO: 3 (Zm-ODP2). The ORFencodes the amino acid sequence set forth in SEQ ID NO: 2 with a singleamino acid alteration (i.e., S37 to T37). The ORF encodes the amino acidsequence set forth in SEQ ID NO: 19. 14 nucleic acid Variant of Zm-ODP2having 91.9% nucleic acid sequence identity to SEQ ID NO: 3 (Zm-ODP2).The ORF encodes the amino acid sequence set forth in SEQ ID NO: 2 with asingle amino acid alteration (i.e., S37 to T37). 15 nucleic acid Variantof Zm-ODP2 having 86.6% nucleic acid sequence identity to SEQ ID NO: 3(Zm-ODP2). The ORF encodes the amino acid sequence set forth in SEQ IDNO: 2 with a single amino acid alteration (i.e., S37 to T37). 16 nucleicacid Variant of Zm-ODP2 having 81.5% nucleic acid sequence identity toSEQ ID NO: 3 (Zm-ODP2). The ORF encodes the amino acid sequence setforth in SEQ ID NO: 2 with a single amino acid alteration (i.e., S37 toT37). 17 nucleic acid Variant of Zm-ODP2 having 75.9% nucleic acidsequence identity to SEQ ID NO: 3 (Zm-ODP2). The ORF encodes the aminoacid sequence set forth in SEQ ID NO: 2 with a single amino acidalteration (i.e., S37 to T37). 18 nucleic acid Variant of Zm-ODP2 having70.4% nucleic acid sequence identity to SEQ ID NO: 3 (Zm-ODP2). The ORFencodes the amino acid sequence set forth in SEQ ID NO: 2 with a singleamino acid alteration (i.e., S37 to T37). 19 Amino acid Variant ofZm-ODP2 having a single amino acid alteration at S37 to T37. 20 Aminoacid Variant of Zm-ODP2 having 97.3% amino acid sequence identity to SEQID NO: 2 (Zm-ODP2). 21 Amino acid Variant of Zm-ODP2 having 92.4% aminoacid sequence identity to SEQ ID NO: 2 (Zm-ODP2). 22 Amino acid Variantof Zm-ODP2 having 87.3% amino acid sequence identity to SEQ ID NO: 2(Zm-ODP2). 23 Amino acid Variant of Zm-ODP2 having 82.4% amino acidsequence identity to SEQ ID NO: 2 (Zm-ODP2). 24 Amino acid Consensussequence of FIG. 2.

Example 4 Agrobacterium-Mediated Transformation

For Agrobacterium-mediated transformation of maize with a plasmidcontaining the Zm-ODP2 operably linked to an oleosin promoter and theselectable marker gene PAT, optimally the method of Zhao is employed(U.S. Pat. No. 5,981,840, and PCT patent publication WO98/32326; thecontents of which are hereby incorporated by reference). Briefly,immature embryos are isolated from maize and the embryos contacted witha suspension of Agrobacerium, where the bacteria are capable oftransferring the ODP2 sequence to at least one cell of at least one ofthe immature embryos (step 1: the infection step). In this step theimmature embryos are optimally immersed in an Agrobacerium suspensionfor the initiation of inoculation. The embryos are co-cultured for atime with the Agrobacerium (step 2: the co-cultivation step). Optimallythe immature embryos are cultured on solid medium following theinfection step. Following this co-cultivation period an optional“resting” step is contemplated. In this resting step, the embryos areincubated in the presence of at least one antibiotic known to inhibitthe growth of Agrobacerium without the addition of a selective agent forplant transformants (step 3: resting step). Optimally the immatureembryos are cultured on solid medium with antibiotic, but without aselecting agent, for elimination of Agrobacerium and for a resting phasefor the infected cells. Next, inoculated embryos are cultured on mediumcontaining a selective agent and growing transformed callus is recovered(step 4: the selection step). Optimally, the immature embryos arecultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus is then regeneratedinto plants (step 5: the regeneration step), and optimally calli grownon selective medium are cultured on solid medium to regenerate theplants.

Example 5 Altering Oil Content and Starch Content of Maize

The full length ODP2 sequence described in Example 1, was used forconstruction of the oleosin driven expression cassette: OLEPRO::ZM-ODP2::NOS TERM. This cassette was inserted into a finaltransformation plasmid using standard protocols. The finaltransformation vector contains OLE PRO::ZM-ODP2::NOS TERM and MO-PATselection marker is transformed into High type-II maize/PHRO3 viaAgrobacerium transformation. Methods of Agrobacerium transformation areoutlined in Example 4.

Transgenic events are recovered and advanced to the greenhouse. Theplants are self-pollinated. At maturity, ears are collected and aportion of seeds (typically 20 kernels from each ear) dissected toseparate the embryo from the endosperm. Dissected seeds are dried downin a lyophilizer overnight. The amount of oil in each embryo isdetermined using NMR. Data for embryo oil %, total embryo oil and embryoweight are collected and analyzed. If changes from High type-IImaize/PHRO3 baseline are observed, a PCR co-segregation analysis isperformed to determine if the changes are correlated with the presenceof transgene (ZM-ODP2).

In addition, germs are also isolated from mature kernels fordetermination of starch and oil concentrations of the seed part.Individual dry seed are soaked overnight at 4° C. in 1 mL of solutioncontaining 20 mM acetate (pH 6.5) and 10 mM mercuric chloride. (Adkinset al. (1966) Starch 7: 213-218). Intact germ is dissected from theseed, dried by lyophilization and recorded for dry weight. Individualgerm is ground for 10 sec in a Silamet amalgam mixer and transferredwith hexane washing into a microcentrifuge tube. The tissue is extractedby stirring with 1 mL of hexane 3×60 min and centrifuged after eachextraction period. The supernatant of extractions is collected andplaced into a preweighed aluminum pan. After evaporation of hexane fromthe weigh pans in a fumehood, final traces of solvent are removed in aforced draft oven at 105° C. for 15 minutes. Cooled weigh pans arereweighed to determine the total weight of oil extracted from the germ.The meal remaining after oil extraction is twice washed with water andcentrifugation (10 min; 1,000×g) and analyzed for starch by a modifiedprocedure for total starch measurement (McCleary et al. (1994) Journalof Cereal Science 20: 51-58). Free sugars are removed by extraction with80% ethanol and the starch dissolved in 90% dimethylsulfoxide. Heatstable α-amylase and high purity amyloglucosidase (very low inβ-glucanse activities) are used to degrade the starch to monomericcarbohydrate. The resulting glucose will be quantitated according to(Jones et al. (1977) Plant Physiol. 60: 379-383) with modification to amicroplate format.

Example 6 Placing ODP2 Sequence Under the Control of a Tissue-PreferredPromoter

The ODP2 gene can be placed under control of an inducible expressionsystem, as described in Zuo et al. (2000) Plant J 24:265-273 and in U.S.Patent Application Publication No. US 2003/0082813 A1, the entirecontents of which are herein incorporated by reference. The G10-90promoter in the XVE vector can be replaced with a tissue-preferredpromoter (e.g. a pollen-, root-stem- or leaf-specific promoter). Avariety of tissue-preferred promoters are well known to those of skillin the art. Because expression of a transgene is activated by thechimeric XVE gene which is controlled by a tissue-preferred promoter inthis Example, the O^(lexA)-46 promoter controlling the ODP2 transgene istherefore tissue-preferred in an inducer-dependent manner. This meansthat ODP2 will be induced only in the presence of an inducer and only inthe specific tissue corresponding to the tissue specific promoter.Appropriate tissues or cell types, can then be collected from thetransgenic plants and used for induction of somatic embryos andregeneration of plants.

Particularly when pollen derived from transgenic plants carrying apollen-specific promoter-XVE/O^(lexA)-46-ODP2 vector is used, progenyplants generated from pollen-derived somatic embryos should be haploidinstead of diploid (see, e.g., Twell et al. (1989) Mol. Gen. Genetics217:240-245 and Twell et al. (1990) Development 109:705-714 forpollen-specific promoters). In this embodiment of the invention, atransgenic plant having in its genome a ODP2 gene under the control ofan inducible, pollen-specific promoter would not normally express thegene. Pollen from such a plant can be cultured in the presence of theinducer until somatic embryogenesis occurs, after which the inducer isremoved and the haploid embryos are permitted to develop into haploidclones according to standard techniques.

Example 7 Generating an Apomictic Plant

Apomixis can be induced by introducing ODP2 into a plant cell in such amanner that the ODP2 gene is expressed in the appropriate tissues (e.g.,nucellus tissue). This can be by means of, but is not limited to,placing the ODP2 gene under the control of a tissue-preferred promoter(e.g., a nucellus-specific promoter), an inducible promoter, or apromoter that is both inducible and tissue-preferred. Inducingexpression of the ODP2 gene, e.g. in the nucellus, producesfertilization-independent embryo formation leading to an apomicticplant. This plant may then be used to establish a true-breeding plantline. Additionally, the vector utilized to transfer ODP2 into the plantcell can include any other desired heterologous gene in addition toODP2, including but not limited to, a marker gene or a gene to confer adesirable trait upon the plant, e.g., a gene resulting in larger plants,faster growth, resistance to stress, etc. This would lead to thedevelopment of an apomictic line with the desired trait.

In a variation of the scheme, plant expression cassettes, including butnot limited to monocot or dicot expression cassettes, directing ODP2expression to the inner integument or nucellus can easily beconstructed. An expression cassette directing expression of the ODP2 DNAsequences to the nucellus can be made using the barley Nuc1 promoter(Doan et al. (1996) Plant Mol Biol. 2:276-284). Such an expression canbe used for plant transformation. Other genes which confer desirabletraits can also be included in the cassette.

It is anticipated that transgenic plants carrying the expressioncassette will then be capable of producing de novo embryos from ODP2expressing nucellar cells. In the case of maize, this is complemented bypollinating the ears to promote normal central cell fertilization andendosperm development. In another variation of this scheme, Nuc1:ODP2transformations could be done using a fie (fertility-independentendosperm)-null genetic background which would promote both de novoembryo development and endosperm development without fertilization (Ohadet al. (1999) The Plant Cell 11:407-416). Upon microscopic examinationof the developing embryos it will be apparent that apomixis has occurredby the presence of embryos budding off the nucellus. In yet anothervariation of this scheme the ODP2 DNA sequences could be delivered asdescribed above into a homozygous zygotic-embryo-lethal genotype. Onlythe adventive embryos produced from somatic nucellus tissue woulddevelop in the seed.

Example 8 Transformation and Regeneration of Maize Embryos

Immature maize embryos from greenhouse donor plants are bombarded with aplasmid containing the ODP2 sequence of the invention operably linked toa promoter. This could be a weak promoter such as nos, a tissue-specificpromoter, such as globulin-1, an inducible promoter such as In2, or astrong promoter such as ubiquitin plus a plasmid containing theselectable marker gene PAT (Wohlleben et al. (1988) Gene 70:25-37) thatconfers resistance to the herbicide Bialaphos. Transformation isperformed as follows.

Maize ears are harvested 8-14 days after pollination and surfacesterilized in 30% Chlorox bleach plus 0.5% Micro detergent for 20minutes, and rinsed two times with sterile water. The immature embryosare excised and placed embryo axis side down (scutellum side up), 25embryos per plate. These are cultured on 560 L medium 4 days prior tobombardment in the dark. Medium 560 L is an N6-based medium containingEriksson's vitamins, thiamine, sucrose, 2,4-D, and silver nitrate. Theday of bombardment, the embryos are transferred to 560Y medium for 4hours and are arranged within the 2.5-cm target zone. Medium 560Y is ahigh osmoticum medium (560 L with high sucrose concentration).

A plasmid vector comprising the ODP2 sequence operably linked to theselected promoter is constructed. This plasmid DNA plus plasmid DNAcontaining a PAT selectable marker is precipitated onto 1.1 μm (averagediameter) tungsten pellets using a CaCl₂ precipitation procedure asfollows: 100 μlprepared tungsten particles in water, 10 μl (1 μg) DNA inTrisEDTA buffer (1 μg total), 100 μl 2.5M CaCl₂, 10 μl 0.1M spermidine.

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 μl 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μlspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

The sample plates are positioned 2 levels below the stooping plate forbombardment in a DuPont Helium Particle Gun. All samples receive asingle shot at 650 PSI, with a total of ten aliquots taken from eachtube of prepared particles/DNA. As a control, embryos are bombarded withDNA containing the PAT selectable marker as described above without thegene of invention.

Following bombardment, the embryos are kept on 560Y medium, an N6 basedmedium, for 2 days, then transferred to 560R selection medium, an N6based medium containing 3 mg/liter Bialaphos, and subcultured every 2weeks. After approximately 10 weeks of selection, bialaphos-resistantcallus clones are sampled for PCR and activity of the gene of interest.In treatments containing the ODP2 gene, it is expected that growth willbe stimulated and transformation frequencies increased, relative to thecontrol. Positive lines are transferred to 288J medium, an MS basedmedium with lower sucrose and hormone levels, to initiate plantregeneration. Following somatic embryo maturation (2-4 weeks),well-developed somatic embryos are transferred to medium for germinationand transferred to the lighted culture room. Approximately 7-10 dayslater, developing plantlets are transferred to medium in tubes for 7-10days until plantlets are well established. Plants are then transferredto inserts in flats (equivalent to 2.5″ pot) containing potting soil andgrown for 1 week in a growth chamber, subsequently grown an additional1-2 weeks in the greenhouse, then transferred to Classic™ 600 pots (1.6gallon) and grown to maturity. Plants are monitored for expression ofthe gene of interest.

Example 9 Ectopic Expression of Maize ODP2 to Induce Embryogenesis

Using the genotype High type II as an example, immature embryos areisolated 15 days after pollination and cultured on 560P medium for 3-5days. At this developmental stage the embryos are too large for callusinitiation under standard culture conditions (see above). Twelve hoursbefore bombardment these embryos are transferred to high osmotic 560Ymedium. Expression cassettes containing the ODP2 cDNA are thenco-introduced into the scutella of these embryos along with anexpression cassette containing genes encoding a screenable markers, suchas green fluorescent protein (GFP) or cyan fluorescent protein (CFP)using methods well described in the art for particle guntransformations. Twelve to 24 hours following bombardment, embryos arethen transferred back to 560P culture medium and incubated in the darkat 26° C. Cultures are then transferred every two weeks untiltransformed colonies appear. It is expected that expression of ODP2 willstimulate adventive embryo formation. This will be apparent when thecultures are compared to controls (transformed without the ODP2 cDNA).Using either inducible expression cassettes, tissue specific promoters,or promoters of varying strengths it will be possible to control thelevels of expression to maximize the formation of adventive embryos.Using either non-responsive genotypes or sub-optimal culture conditionswith responsive genotypes, only the transformed cells expressing theODP2 cDNA will form embryos and regenerate plants. In this manner,ODP2-induced embryo proliferation can be used as a positive selectivemarker (only the cells expressing the gene will form embryos) andtransformants can be recovered without a negative selective agent (i.e.bialaphos, basta, kanamycin, etc.).

Example 10 Ectopic Expression of Maize ODP2 is Sufficient to StimulateOrganogenesis/Embryogenesis and Increases Transformation Frequencies inRecalicitrant Tissues

There exists only a small developmental window in which maize embryosare amenable to tissue culture growth, a prerequisite fortransformation. Normally this occurs between 9-12 days after pollinationwhen the immature embryos are between 1.0-1.5 mm in length. Older,larger embryos fail to produce embryogenic callus and thus cannot betransformed. To demonstrate that ODP2 can be used to induceembryogenesis, embryos from the maize inbred PH581, ATCC depositPTA-4432, were isolated 17 days after pollination and used fortransformation experiments. Isolated embryos were cultured on 605Jmedium (a medium containing both full strength MS salts (macro andmicronutient) and 0.6×N6 macronutrient salts plus additional B5micronutrients, with a mixture of SH and Eriksson's vitamin, L-prolineand casamino acids, silver nitrate, 0.3 mg/l 2,4-D and 1.2 mg/l Dicamba,2% sucrose and 0.06% glucose, solidified with agar). The embryos wereincubated in the dark at 28° C. overnight. Embryos were shot in a methodsimilar to that in Example 8 substituting 0.6 μm gold particles fortungsten. DNA was delivered using co-transformation, as noted above. Asa control, embryos were shot with a 1:1 mixture of plasmid DNA'scontaining a Ubiquitin driven yellow fluorescence protein (YFP) and aplasmid containing a Ubiquitin driven uidA gene (GUS). In the ODP2treatment the embryos were bombarded with a 1:1 mixture of plasmid DNA'scontaining the Ubiquitin promoter driving expression of YFP (Ubi:YFP)and a plasmid containing ODP2 (SEQ ID NO: 3) driven by the maizeUbiquitin promoter (Ubi:ODP2). Each treatment contained 20 embryos.After one month of culture the embryos were observed under thedissecting microscope using epifluorescence.

As mentioned above, it is well known in the art that there is a narrowwindow in embryo ontogeny where embryos are culture/transformationresponsive and this window occurs when embryos are in 1-2 mm in lengthwhich is typically 9-12 days after pollination. Since these embryos weretaken at 17 days after pollination no multicellular colonies wereexpected in the control treatment. As expected, hundreds of cellstransiently expressing the YFP protein were visible under a fluorescentmicroscope in the control treatment, and in this population offluorescing cells, cell division was very rare. Cells transientlyexpressing YFP were also apparent in the ODP2 treatment. However, in theODP2 treatment, cell division was apparent in all of the bombardedembryos with up to 50 multicellular colonies observed per embryo (datanot shown). No events were observed in the control treatment while 100%of the ODP2 embryos were transformed with 5-50 events/embryo. Embryomorphology was clearly visible in many of these growing transgeniccolonies.

As mentioned above ODP2 expression was sufficient to induceembryogenesis in larger and normally non-responsive embryos. In asimilar manner, controlled ODP2 expression should allow transformationof other vegetative tissues such as leaves, stems, and even seed. ODP2driven by the ubiquitin promoter was used to transform stem tissues.Transformed embryos were recovered from stem tissues (data not shown).

Example 11 Transient Expression of the ODP2 Gene Product to InduceEmbryogenesis

It may be desirable to “kick start” meristem formation by transientlyexpressing the ODP2 gene product. This can be done by delivering ODP2 5′capped polyadenylated RNA, expression cassettes containing ODP2 DNA, orODP2 protein. All of these molecules can be delivered using a biolisticsparticle gun. For example, 5′ capped polyadenylated ODP2 RNA can easilybe made in vitro using Ambion's mMes sage mMachine kit. Following adelivery procedure outlined above, RNA is co-delivered along with DNAcontaining an agronomically useful expression cassette. It is expectedthat cells receiving ODP2 will form embryos and a large portion of thesewill have integrated the agronomic gene. Plants regenerated from theseembryos can then be screened for the presence of the agronomic gene.

Example 12 Modifying the Regenerative Capacity of a Plant

To demonstrate that ODP2 improves the regenerative capacity of maizetissues transformants were produced in the genotype High Type II withconstructs containing the ODP2 gene driven by the maize Oleosinpromoter. The Oleosin promoter is highly specific and is expressed onlyin scutella of developing embryos. Transformants were produced usingboth particle gun (as described in example 4 above) and Agrobacerium(U.S. Pat. No. 5,981,840). Putative transformants were grown in thegreenhouse and were completely normal in phenotype. Ears were pollinatedand segregating embryos were isolated from a particle gun event at 17DAP (days after pollination) and from Agrobacerium derived events at 24DAP. Embryos cultured at such late stages would be expected to germinateon regeneration medium. This was observed in the wild-type segregatesbut germination was delayed in the transformed embryos. In addition todelayed germination, somatic embryos proliferated from the scutella ofthe transformed embryos (data not shown) when cultured on regenerationmedium. The maize Oleosin promoter is highly expressed at these latestages of development and this result demonstrates that the maize ODP2gene is sufficient to induce embryogenesis in a normally non-responsivetissue.

Example 13 Transient Expression of ODP2 Enhances Transformation

Parameters of the transformation protocol can be modified to insure thatthe increased ODP2 activity is transient. One such method involvesprecipitating the ODP2-containing plasmid in a manner that precludessubsequent release of the DNA (thus, transcription from theparticle-bound DNA can occur, but the frequency with which its releasedto become integrated into the genome is greatly reduced. Such aprecipitation relies on the chemical PEI, and it could be used asdiscussed below.

The ODP2 plasmid is precipitated onto gold particles with PEI, while thetransgenic expression cassette (UBI::moPAT˜GFPm::pinII) to be integratedis precipitated onto gold particles using the standard Ca⁺⁺ method.Briefly, coating gold particles with PEI is done as follows. First, thegold particles are washed. Thirty-five mg of gold particles, for example1.0 μM in average diameter (A.S.I. #162-0010), are weighed out in amicrocentrifuge tube, and 1.2 ml absolute EtOH is added and vortexed forone minute. The tube is set aside for 15 minutes at room temperature andthen centrifuged at high speed using a microfuge for 15 minutes at 4° C.The supernatant is discarded and a fresh 1.2 ml aliquot of EtOH isadded, vortexed for one minute, centrifuged for one minute and thesupernatant again discarded (this is repeated twice). A fresh 1.2 mlaliquot of EtOH is added, and this suspension (gold particles in EtOH)can be stored at −20° C. for weeks. To coat particles withpolyethylimine (PEI; Sigma #P3143), start with 250 μl of washed goldparticle/EtOH, centrifuge and discard EtOH. Wash once in 100 μl ddH2O toremove residual ethanol. Add 250 μl of 0.25 mM PEI, pulse-sonicate tosuspend particles and then plunge tube into dry ice/EtOH bath toflash-freeze suspension into place. Lyophilize overnight. At this point,dry, coated particles can be stored at −80° C. for at least 3 weeks.Before use, rinse particles 3 times with 250 μl aliquots of 2.5 mM HEPESbuffer, ph 7.1, with 1× pulse-sonication and then quick vortex beforeeach centrifugation. Suspend in final volume of 250 μl HEPES buffer.Aliquot 25 μl to fresh tubes before attaching DNA. To attach uncoatedDNA, pulse-sonicate the particles, then add DNA's and mix by pipettingup and down a few times with a Pipetteman™. Let sit for at least 2minutes, spin briefly (i.e. 10 seconds), remove supernatant and add 60μl EtOH. Spot onto macrocarriers and bombard following standardprotocol. The Ca⁺⁺ precipitation and bombardment follows standardprotocol for the PDS-1000.

The two particle preparations are mixed together; and the mixture isbombarded into scutellar cells on the surface of immature embryos (somecells receiving only an ODP2 particle, some cells receiving only aPAT˜GFP particle and some cells receiving both). PEI-mediatedprecipitation results in a high frequency of transiently expressingcells on the surface of the immature embryo and extremely lowfrequencies of recovery of stable transformants (relative to the Ca⁺⁺method). Thus, the PEI-precipitated ODP2 cassette expresses transientlyand stimulates a burst of embryogenic growth on the bombarded surface ofthe tissue (i.e. the scutellar surface), but this plasmid does notintegrate. The PAT˜GFP plasmid released from the Ca⁺⁺/gold particlesintegrates and expresses the selectable marker at a frequency thatresult in substantially improved recovery of transgenic events.

As a control treatment, PEI-precipitated particles containing aUBI::GUS::pinII (instead of ODP2) are mixed with the PAT˜GFP/Ca⁺⁺particles. Immature embryos from both treatments are moved onto culturemedium containing 3 mg/l bialaphos. After 6-8 weeks, GFP+,bialaphos-resistant calli are observed in the PEI/ODP2 treatment at amuch higher frequency relative to the control treatment (PEI/GUS).

The ODP2 plasmid is precipitated onto gold particles with PEI, and thenintroduced into scutellar cells on the surface of immature embryos, andsubsequent transient expression of the ODP2 gene elicits a rapidproliferation of embryogenic growth. During this period of inducedgrowth, the explants are treated with Agrobacerium using standardmethods for maize (Zhao et al., U.S. Pat. No. 5,981,840), with T-DNAdelivery into the cell introducing a transgenic expression cassette suchas UBI::moPAT˜GFPm::pinII. After co-cultivation, explants are allowed torecover on normal culture medium, and then are moved onto culture mediumcontaining 3 mg/l bialaphos. After 6-8 weeks, GFP⁺, bialaphos-resistantcalli are observed in the PEI/ODP2 treatment at a much higher frequencyrelative to the control treatment (PEI/GUS).

Example 14 Transient Expression of the ODP2 Polynucleotide Product toInduce Somatic Embryogenesis

It may be desirable to “kick start” somatic embryogenesis by transientlyexpressing the ODP2 polynucleotide product. This can be done bydelivering ODP2 5′capped polyadenylated RNA, expression cassettescontaining ODP2 DNA, or ODP2 protein. All of these molecules can bedelivered using a biolistics particle gun. For example 5′cappedpolyadenylated ODP2 RNA can easily be made in vitro using Ambion'smMessage mMachine kit. Following the procedure outline above RNA isco-delivered along with DNA containing an agronomically usefulexpression cassette, and a marker used for selection/screening such asUbi::moPAT˜GFPm::pinll. The cells receiving the RNA will immediatelyform somatic embryos and a large portion of these will have integratedthe agronomic gene, and these can further be validated as beingtransgenic clonal colonies because they will also express the PAT˜GFPfusion protein (and thus will display green fluorescence underappropriate illumination). Plants regenerated from these embryos canthen be screened for the presence of the agronomic gene.

Example 15 Ectopic Expression of ODP2 in Early Zygotic Embryos IncreasesSeed Set During Abiotic Stress Episodes

During periods of abiotic stress such as during a drought episode,embryo development often is halted resulting in aborted kernels on theear. Preventing this kernel loss will increase or maintain yield. Toincrease seed set during periods of abiotic stress, the ODP2 gene iscloned into an expression cassette behind an early-embryo promoter suchas LEC1, and this expression cassette is cloned along with aselectable/screenable marker into an Agrobacerium T-DNA region. Forexample, the following T-DNA is constructed:RB-LEC1::ODP2::pinII/Ubi::moPAT˜GFPm::pinII-LB. This T-DNA is introducedinto a maize inbred using standard Agrobacerium transformation methods.Transgenic plants are screened for single-copy integrations, and thenplanted in individual pots in the greenhouse. Transgenic plants areselfed and out-crossed to wild-type plants. Plants transgenic for theODP2 expression cassette are easily tracked (using the cosegregatingmarker) through either BASTA resistance or green fluorescence conferredby the PAT˜GFP fusion protein. Transgenic plants are planted in thefield, and subjected to various degrees of drought stress duringflowering and seed-set. Under identical stress regimes, the transgenicplants have much higher numbers of developed kernels relative towild-type (non-transgenic) plants.

Example 16 Expression of ODP2 in Double-Haploid Production

There are two necessary steps in the production of double-haploidgermplasm from maize inbreds. The first is induction of embryogenesisfrom a haploid cell, and the second is chromosome doubling to convertthe haploid to a doubled-haploid.

The ODP2 gene can be used to generate haploid plants at high frequencies(i.e. improving the efficiency of step one of the process). Variousstrategies for accomplishing this are described below.

A. The following expression cassettes are placed in between a single setof T-DNA borders. T-DNA cassette #1 comprisesRB-loxP/gal::FLP::pinII/PG47::C1-GAL-EcR::pinII/Ubi::PAT::pinII/Ubi:frt:YFP::pinII:frt:ODP2::pinII/LEC1::Cre::pinII/loxP-LB.To use this construct, it is first transformed into a maize genotypeusing Agrobacerium methods for 2-T-DNA transformation into immatureembryos (Miller et al. (2002) Transgene Research 11:381-96).

In addition to the T-DNA diagramed above, this method also introducesT-DNA cassette #2 containing RB-Ole::WUS2::pinII/Ubi::CFP::pinII-LB, butwhich integrates at an unlinked location in the genome. T-DNA cassette#2 provides a means of recovering transformed events without chemicalselection and then later segregating the T-DNA cassette #2 away from #1.Standard tissue culture and regeneration methods are used.

Transgenic plants are grown until the microspores in the developingtassel are at the uninucleate stage. At this point, the tassel isexcised and pretreated by wrapping in moist paper towel and incubatedfor 14-17 days at 8-10° C. Following pre-treatment, tassels are surfacesterilized by soaking for 10 minutes in sodium hypochlorite solution(i.e. 50% Chlorox), and then rinsed twice in sterile water. The anthersare then excised from the tassel and placed on solid anther culturemedium using standard media formulations developed for maize antherculture (see Petolino and Genovesi (1994) in The Maize Handbook, (Walbotand Freeling, eds), pages 701-704). Once the anthers are on solidmedium, the inducing agent methoxifenozide is pipetted directly onto thesolid medium (for example, a 10 mM stock of methoxyfenozide is dilutedto 10 uM by pipetting 30 ul of the stock onto the surface of 30 ml ofsolid medium and allowed to equilibrate before adding plant tissue).This will induce expression of FLP recombinase in the uninucleatemicrospores in the anther. FLP activity would excise the YFP gene,functionally linking the strong Ubiquitin promoter with the ODP2 gene.This burst of ODP2 expression will induce embryogenesis at highfrequencies in the haploid uninucleate microspores. After the embryosbegin developing, the embryogenic-specific promoter LEC1 will turn onCre expression and this recombinase will excise the entire transgenecassette. Excision of the expression cassette and the concomitant lossof ODP2 expression will permit embryo maturation and subsequent plantregeneration to occur. During embryo development stimulated by theprocess described above, colchicine can be added (i.e. a 0.01 to 1.0%solution) to induce chromosome doubling. Doubled haploid plants arerecovered that no longer contain T-DNA cassette #2 (because it wassegregated away) and only contain the RB-loxP-LB sequence left behindafter excision of almost all of cassette #1.

An alternative way to accomplish the above scenario would be to placethe ODP2 gene behind a promoter that is active during microsporedevelopment. For example the maize promoters PG47, Zm-POL67 and Zm-POL95are all promoters active during microspore development. In transgenicplants containing the PG47::ODP2 expression cassette, embryo formationis initiated in the microspores of the developing tassel. Anembryo-specific promoter such as LEC1 or Glb1 is then used to driveexpression of the Cre gene, which excises the loxP-flanked ODP2 and Creexpression cassettes. These embryos are then capable of maturing andgerminating into haploid plants, or if exposed to a doubling agent suchas colchicines, double-haploid plats are generated.

Example 17 ODP2 Expression for Positive Selection

It is expected that transformants can be recovered using ODP2 expressionto provide a positive selection means under reduced auxin levels or inthe absence of auxins in the medium, and in the absence of herbicide orantibiotic selection.

To determine if ODP2 can be used in a positive selection scheme,transformation experiments, using any standard method including particlegun or Agrobacerium, can be performed. Transformants are selected onmedium with normal auxin levels, or on medium with reduced or no auxin,or visually (using GFP) on medium without bialaphos. Transformationfrequencies are based on numbers of embryos with one or moremulticellular GFP positive cell clusters. For example, one can test thisconcept using two treatment variables. The first is that immatureembryos are bombarded with a control plasmid (UBI:PAT˜GFP) or withUBI:PAT˜GFP+In2:ODP2. The second variable is that the bombarded embryosare divided onto either normal bialaphos-containing selection medium(with normal auxin levels of 2 mg/L 2,4-D), or medium with no bialaphosand reduced 2,4-D levels (0.5 mg/L). It is expected from previousstudies of positive selection that on bialaphos selection the ODP2treatment will result in higher transformation frequency than thecontrol. It is also anticipated that the low auxin medium (0.5 mg/L2,4-D) will result in reduced growth rates. Consistent with this, it isexpected that for the control plasmid treatment (UBI:PAT˜GFP), recoveryof GFP-expressing (fluorescent) colonies will be reduced relative tohighly effective bialaphos selection treatment. In contrast, it isexpected that ODP2 expression, through its stimulation of embryogenesis,may compensate for the low auxin environment, providing a growthadvantage to the transgenic colonies, and maintaining the efficiency oftransformant recovery at approximately the same range as theODP2/bialaphos-selected treatment.

On medium completely devoid of auxin, it is expected that colonies willonly be observed in the ODP2 treatment. In this experiment, immatureembryos are transformed with either the control plasmid (UBI:PAT˜GFP) orwith UBI:PAT˜GFP+In2:ODP2, and then plated either onto 3.0 mg/Lbialaphos, 2.0 mg/L 2,4-D medium or onto no-bialaphos, no 2,4-D medium(in this latter treatment, wild-type maize callus will not exhibitembryonic growth). Again, it is expected that expression of the ODP2polynucleotide will increase transformation significantly over thecontrol plasmid value on normal auxin-containing, bialaphos selectionmedium. Also, it is expected that no transformants will be recoveredwith the control plasmid on medium devoid of exogenous auxin.

Even on auxin-containing medium, the ODP2 polynucleotide in combinationwith GFP+ expression can be used to recover transformants withoutchemical selection. For example, under these conditions it is expectedthat the recovery of transformants will be relatively efficient, but mayrequire more diligence than the low- or no-auxin treatments above toseparate the GFP-expressing colonies from the growing callus population.

Example 18 Soybean Embryo Transformation

Soybean embryos are bombarded with a plasmid containing the ODP2sequence operably linked to a promoter. This could be a weak promotersuch as nos, a tissue-specific promoter, such as globulin-1, aninducible promoter such as In2, or a strong promoter such as ubiquitinplus a plasmid containing the selectable marker gene PAT (Wohlleben etal. (1988) Gene 70:25-37) that confers resistance to the herbicideBialaphos. Transformation is performed as follows.

To induce somatic embryos, cotyledons, 3-5 mm in length dissected fromsurface-sterilized, immature seeds of the soybean cultivar A2872, arecultured in the light or dark at 26° C. on an appropriate agar mediumfor six to ten weeks. Somatic embryos producing secondary embryos arethen excised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos that multiplied as early,globular-staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can maintained in 35 ml liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 ml of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A Du Pont Biolistic PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene that can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188), and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The expression cassette comprising the ODP2 operably linkedto the promoter can be isolated as a restriction fragment. This fragmentcan then be inserted into a unique restriction site of the vectorcarrying the marker gene.

To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (inorder): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μl 70% ethanol andresuspended in 40 μl of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi, and the chamber is evacuated to a vacuum of 28inches mercury. The tissue is placed approximately 3.5 inches away fromthe retaining screen and bombarded three times. Following bombardment,the tissue can be divided in half and placed back into liquid andcultured as described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post-bombardment with freshmedia containing 50 mg/ml hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post-bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 19 Sunflower Meristem Tissue Transformation Prophetic Example

Sunflower meristem tissues are transformed with an expression cassettecontaining the ODP2 sequence operably linked to a promoter. This couldbe a weak promoter such as nos, a tissue-specific promoter, such asglobulin-1, an inducible promoter such as In2, or a strong promoter suchas ubiquitin plus a plasmid containing the selectable marker gene PAT(Wohlleben et al. (1988) Gene 70:25-37) that confers resistance to theherbicide Bialaphos. Transformation is performed as follows. See alsoEuropean Patent Number EP 0 486233, herein incorporated by reference,and Malone-Schoneberg et al. (1994) Plant Science 103:199-207).

Mature sunflower seed (Helianthus annuus L.) are dehulled using a singlewheat-head thresher. Seeds are surface sterilized for 30 minutes in a20% Clorox bleach solution with the addition of two drops of Tween 20per 50 ml of solution. The seeds are rinsed twice with sterile distilledwater.

Split embryonic axis explants are prepared by a modification ofprocedures described by Schrammeijer et al. (Schrammeijer et al. (1990)Plant Cell Rep. 9:55-60). Seeds are imbibed in distilled water for 60minutes following the surface sterilization procedure. The cotyledons ofeach seed are then broken off, producing a clean fracture at the planeof the embryonic axis. Following excision of the root tip, the explantsare bisected longitudinally between the primordial leaves. The twohalves are placed, cut surface up, on GBA medium consisting of Murashigeand Skoog mineral elements (Murashige et al. (1962) Physiol. Plant., 15:473-497), Shepard's vitamin additions (Shepard (1980) in EmergentTechniques for the Genetic Improvement of Crops (University of MinnesotaPress, St. Paul, Minn.), 40 mg/l adenine sulfate, 30 g/l sucrose, 0.5mg/l 6-benzyl-aminopurine (BAP), 0.25 mg/l indole-3-acetic acid (IAA),0.1 mg/l gibberellic acid (GA₃), pH 5.6, and 8 g/l Phytagar.

The explants are subjected to microprojectile bombardment prior toAgrobacerium treatment (Bidney et al. (1992) Plant Mol. Biol.18:301-313). Thirty to forty explants are placed in a circle at thecenter of a 60×20 mm plate for this treatment. Approximately 4.7 mg of1.8 mm tungsten microprojectiles are resuspended in 25 ml of sterile TEbuffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0) and 1.5 ml aliquots are usedper bombardment. Each plate is bombarded twice through a 150 mm nytexscreen placed 2 cm above the samples in a PDS 1000® particleacceleration device.

Disarmed Agrobacterium tumefaciens strain EHA105 is used in alltransformation experiments. A binary plasmid vector comprising theexpression cassette that contains the ODP2 gene operably linked to thepromoter is introduced into Agrobacerium strain EHA105 viafreeze-thawing as described by Holsters et al. (1978) Mol. Gen. Genet.163:181-187. This plasmid further comprises a kanamycin selectablemarker gene (i.e., nptII). Bacteria for plant transformation experimentsare grown overnight (28° C. and 100 RPM continuous agitation) in liquidYEP medium (10 gm/l yeast extract, 10 gm/l Bactopeptone, and 5 gm/lNaCl, pH 7.0) with the appropriate antibiotics required for bacterialstrain and binary plasmid maintenance. The suspension is used when itreaches an OD₆₀₀ of about 0.4 to 0.8. The Agrobacerium cells arepelleted and resuspended at a final OD600 of 0.5 in an inoculationmedium comprised of 12.5 mM MES pH 5.7, 1 gm/l NH₄Cl, and 0.3 gm/lMgSO₄.

Freshly bombarded explants are placed in an Agrobacerium suspension,mixed, and left undisturbed for 30 minutes. The explants are thentransferred to GBA medium and co-cultivated, cut surface down, at 26° C.and 18-hour days. After three days of co-cultivation, the explants aretransferred to 374B (GBA medium lacking growth regulators and a reducedsucrose level of 1%) supplemented with 250 mg/l cefotaxime and 50 mg/lkanamycin sulfate. The explants are cultured for two to five weeks onselection and then transferred to fresh 374B medium lacking kanamycinfor one to two weeks of continued development. Explants withdifferentiating, antibiotic-resistant areas of growth that have notproduced shoots suitable for excision are transferred to GBA mediumcontaining 250 mg/l cefotaxime for a second 3-day phytohormonetreatment. Leaf samples from green, kanamycin-resistant shoots areassayed for the presence of NPTII by ELISA and for the presence oftransgene expression by assaying for ODP2 activity.

NPTII-positive shoots are grafted to Pioneer® hybrid 6440 in vitro-grownsunflower seedling rootstock. Surface sterilized seeds are germinated in48-0 medium (half-strength Murashige and Skoog salts, 0.5% sucrose, 0.3%gelrite, pH 5.6) and grown under conditions described for explantculture. The upper portion of the seedling is removed, a 1 cm verticalslice is made in the hypocotyl, and the transformed shoot inserted intothe cut. The entire area is wrapped with parafilm to secure the shoot.Grafted plants can be transferred to soil following one week of in vitroculture. Grafts in soil are maintained under high humidity conditionsfollowed by a slow acclimatization to the greenhouse environment.Transformed sectors of T₀ plants (parental generation) maturing in thegreenhouse are identified by NPTII ELISA and/or by ODP2 activityanalysis of leaf extracts while transgenic seeds harvested fromNPTII-positive T₀ plants are identified by ODP2 activity analysis ofsmall portions of dry seed cotyledon.

An alternative sunflower transformation protocol allows the recovery oftransgenic progeny without the use of chemical selection pressure. Seedsare dehulled and surface-sterilized for 20 minutes in a 20% Cloroxbleach solution with the addition of two to three drops of Tween 20 per100 ml of solution, then rinsed three times with distilled water.Sterilized seeds are imbibed in the dark at 26° C. for 20 hours onfilter paper moistened with water. The cotyledons and root radical areremoved, and the meristem explants are cultured on 374E (GBA mediumconsisting of MS salts, Shepard vitamins, 40 mg/l adenine sulfate, 3%sucrose, 0.5 mg/l 6-BAP, 0.25 mg/l IAA, 0.1 mg/l GA, and 0.8% Phytagarat pH 5.6) for 24 hours under the dark. The primary leaves are removedto expose the apical meristem, around 40 explants are placed with theapical dome facing upward in a 2 cm circle in the center of 374M (GBAmedium with 1.2% Phytagar), and then cultured on the medium for 24 hoursin the dark.

Approximately 18.8 mg of 1.8 μm tungsten particles are resuspended in150 μl absolute ethanol. After sonication, 8 μl of it is dropped on thecenter of the surface of macrocarrier. Each plate is bombarded twicewith 650 psi rupture discs in the first shelf at 26 mm of Hg helium gunvacuum.

The plasmid of interest is introduced into Agrobacterium tumefaciensstrain EHA105 via freeze thawing as described previously. The pellet ofovernight-grown bacteria at 28° C. in a liquid YEP medium (10 g/l yeastextract, 10 g/l Bactopeptone, and 5 g/l NaCl, pH 7.0) in the presence of50 μg/l kanamycin is resuspended in an inoculation medium (12.5 mM 2-mM2-(N-morpholino) ethanesulfonic acid, MES, 1 g/l NH₄Cl and 0.3 g/l MgSO₄at pH 5.7) to reach a final concentration of 4.0 at OD 600.Particle-bombarded explants are transferred to GBA medium (374E), and adroplet of bacteria suspension is placed directly onto the top of themeristem. The explants are co-cultivated on the medium for 4 days, afterwhich the explants are transferred to 374C medium (GBA with 1% sucroseand no BAP, IAA, GA3 and supplemented with 250 μg/ml cefotaxime). Theplantlets are cultured on the medium for about two weeks under 16-hourday and 26° C. incubation conditions.

Explants (around 2 cm long) from two weeks of culture in 374C medium arescreened for ODP2 activity using assays known in the art. After positive(i.e., for ODP2 expression) explants are identified, those shoots thatfail to exhibit ODP2 activity are discarded, and every positive explantis subdivided into nodal explants. One nodal explant contains at leastone potential node. The nodal segments are cultured on GBA medium forthree to four days to promote the formation of auxiliary buds from eachnode. Then they are transferred to 374C medium and allowed to developfor an additional four weeks. Developing buds are separated and culturedfor an additional four weeks on 374C medium. Pooled leaf samples fromeach newly recovered shoot are screened again by the appropriate proteinactivity assay. At this time, the positive shoots recovered from asingle node will generally have been enriched in the transgenic sectordetected in the initial assay prior to nodal culture.

Recovered shoots positive for ODP2 expression are grafted to Pioneerhybrid 6440 in vitro-grown sunflower seedling rootstock. The rootstocksare prepared in the following manner. Seeds are dehulled andsurface-sterilized for 20 minutes in a 20% Clorox bleach solution withthe addition of two to three drops of Tween 20 per 100 ml of solution,and are rinsed three times with distilled water. The sterilized seedsare germinated on the filter moistened with water for three days, thenthey are transferred into 48 medium (half-strength MS salt, 0.5%sucrose, 0.3% gelrite pH 5.0) and grown at 26° C. under the dark forthree days, then incubated at 16-hour-day culture conditions. The upperportion of selected seedling is removed, a vertical slice is made ineach hypocotyl, and a transformed shoot is inserted into a V-cut. Thecut area is wrapped with parafilm. After one week of culture on themedium, grafted plants are transferred to soil. In the first two weeks,they are maintained under high humidity conditions to acclimatize to agreenhouse environment.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed:
 1. A method of promoting embryogenesis in amicrospore culture or an anther culture, wherein said method comprisesintroducing into a plant or a plant cell an OVULE DEVELOPMENT PROTEIN 2(ODP2) nucleotide sequence that encodes an ODP2 polypeptide comprisingan amino acid sequence selected from the group consisting of: (a) theamino acid sequence of SEQ ID NO: 2; and (b) an amino acid sequencehaving at least 85% sequence identity to SEQ ID NO: 2, wherein saidpolypeptide comprises two APETALA2 (AP2) domains and wherein thepolypeptide has ODP2 activity rendering the microspore culture or theanther culture embryogenic; and, thereby inducing embryogenesis in themicrospore culture or the anther culture; and selecting an embryogenicmicrospore or embryogenic anther culture.
 2. The method of claim 1,wherein said ODP2 nucleotide sequence is under the control of a tissuespecific promoter that is active in a microspore.
 3. The method of claim1, wherein said ODP2 nucleotide sequence is under the control of apromoter that is active during microspore development.
 4. The method ofclaim 3, wherein said promoter that is active during microsporedevelopment is the maize PG47 promoter or the zm-G13 promoter.
 5. Themethod of claim 1, wherein the ODP2 nucleotide sequence is under thecontrol of an inducible promoter.
 6. The method of claim 1, wherein theODP2 nucleotide sequence is under the control of a promoter that is aninducible pollen-specific promoter.